User talk:Kuthuparamba

Estimation of Homocysteine in Serum
 Purpose of the examination Homocysteine assay intended for the ELISA based quantitation of L-homocysteine in serum or plasma.

It helps in the diagnosis and treatment of patients suspected to have homocystinuria / hyperhomocysteinemia.

Principle of the procedure used for examinations; Solid phase Enzyme immunoassay (competitive assay) is used for the determination of Homocysteine in blood. Protein-bound Homocysteine is reduced to free Homocysteine, and is enzymatically converted to S Adenosyl L Homocysteine (SAH) in a separate procedure prior to immunoassay. Enzyme is specific for L Homocysteine, which is the predominant (only) form present in blood.

Dithiothreitol reduces Homocysteine, mixed disulphide and protein-bound forms of Homocysteine to free Homocysteine.

This Homocysteine is converted to S Adenosyl L Homocysteine by the use of SAH hydrolase and excess adenosine.

Test is based on competition between SAH in sample and immobilized SAH bound to wells of microtiter plates for binding sites on a monoclonal anti-SAH antibody. After removal of unbound anti-SAH antibody, secondary rabbit anti-mouse antibody labeled with HRP is added. Peroxidase activity is measured spectrophotometrically after addition of substrate. Absorbance is inversely related to concentration of Homocysteine in the sample. Primary sample collection (e.g. plasma, serum, urine); EDTA-plasma or serum. Serum samples should be allowed to clot for no more than 30 min before separation. Should be kept on ice prior.

EDTA plasma samples should also be kept on ice prior to centrifugation.

Type of container and additives; Serum 5 ml

Required equipment and reagents; Homocysteine controls, pipettes, 37 C incubator, ELISA reader. Reagent A (Assay buffer) – Phosphate buffer, <0.1% sodium azide Reagent B (Adenosine/DTT) – Adenosine, DTT, citric acid Reagent C (SAH hydrolase) Reagent D (Enzyme inhibitor) - <0.2% Thimerosal, phosphate buffer Reagent E (Adenosine deaminase) Reagent F – a SAH antibody Reagent G – Enzyme conjugate Reagent H – Substrate Reagent S – Stop solution, Wash concentrate, Calibrators Calibration procedures (metrological traceability);

Procedural steps; Sample pre-treatment solution is made up no more than 1 hr prior to the start of the assay. Calibrators and samples are diluted in glass tubes in the ratio 25 µl sample/calibrator/control + 500 µl sample pre-treatment solution and mixed well. Incubated for 30’ at 37 C. After cooling, 500 µl Reagent D is added, mixed and incubated for 15’ at 18-25 C. 500 µl Reagent E is added and incubated for 5’ at 18-25 C.

Pipette out 25 µl diluted calibrator/sample/control from earlier step into wells of SAH coated microtitre strips. 200 µl Reagent F added to each well and incubated for 30’ at 18-25 C. After washing, empty the wells on paper towels. 100 µl Reagent G is added to each well and incubated for 20’ at 18-25 C. After washing, empty the wells on paper towels. 100 µl Reagent H is added to each well and incubated for 10’ at 18-25 C. 100 µl Reagent S is added to each well. Shake and read at 450 nm within 15’. Quality control procedures; Method is checked and calibrated at regular intervals to check the linearity of the method with known standards and calibrators.

Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Bilirubin, hemoglobin, triglycerides, RBC, protein and sodium fluoride can have interference.

Protein rich food gives high Homocysteine levels and should be avoided late in the day before sampling.

Drugs involving S Adenosyl Methionine can give false high values.

Pateints who have received mouse monoclonal antibodies for therapy or diagnosis can have interference on assay.

Methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants and 6 azauridine can increase Homocysteine levels.

Cross-reactivity is also seen with Adenosine, cystathionine, L Cysteine, glutathione and thiolactones.

Biological reference intervals; 5.0 – 15.0 µmol/L.

Reportable interval of examination results. 2-5 days.

Alert/critical values, where appropriate, Any value above 25 µmol/L is alerted to the clinician immediately.

Laboratory interpretation; Values <15 µmol/L are considered normal. Moderate hyperhomocysteinemia – 16 – 30 µmol/L. Intermediate – 31 – 100 µmol/L. Severe - > 100 µmol/L.

Safety precautions; All samples and reagents are handled with utmost care. Samples are disposed following standard disposal protocols.

Potential sources of variability. Bilirubin, hemoglobin, triglycerides, RBC, protein and sodium fluoride can have interference. Protein rich food gives high Homocysteine levels and should be avoided late in the day before sampling.

Estimation of Adenosine Deaminase in Pleural Fluid in Ascitic Fluid in Pericardial Fluid
Purpose of the examination Determination of Adenosine deaminase activity in serum, plasma and biological fluids. ADA is an enzyme widely distributed in mammalian tissues, particularly in T lymphocytes. Increased levels of ADA are found in various forms of tuberculosis making it a marker for the same. Principle of the procedure used for examinations; ADA hydrolyzes adenosine to ammonia and inosine. Ammonia formed further reacts with phenol and hypochlorite to form a blue indophenol complex with sodium nitroprusside acting as a catalyst. Intensity of the blue color is directly proportional to amount of ADA present in the sample. Adenosine + Water  Ammonia + Inosine Ammonia + Phenol + Hypochlorite  Blue indophenol complex Primary sample collection (e.g. plasma, serum, urine); Pleural fluid Type of container and additives; Clean plain container, no preservatives Required equipment and reagents; ELISA reader, kit reagents supplied by manufacturers Calibration procedures (metrological traceability); Procedure and protocol are checked at regular intervals to assess the quality of assay. Procedural steps; All reagents and samples are brought to room temperature. Working phenol reagent and hypochlorite reagent are prepared. Spectrophotometer filter is set at 570 – 630 nm.

Blank	Standard	Standard Blank	Test Buffer reagent	0.20	0.20	--	-- Adenosine reagent	--	--	0.20	0.20 Deionized water	0.20	--	--	-- Standard	--	0.20	--	-- Sample	--	--	--	0.20

Mix well and incubate at 37 C for exactly 60 min, and then add the following reagents. Blank	Standard	Standard Blank	Test Working phenol reagent	1.00	1.00	1.00	       1.00 Sample	--	--	0.02	-- Working hypo-Chlorite reagent	1.00	1.00	1.00	1.00

Mixed well and incubated at 37 C for 15’ or at RT for 30’. Absorbance of Blank (AB), Sample Blank (ASB), Standard (AS) and Test (AT) measured against distilled water. AT - ASB Total ADA activity in U/L 	=		x 50 AS - AB Quality control procedures; For each lot, standard control and standard blank are run, which are validated at regular intervals. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Lipemic and hemolysed samples are avoided as they can affect the results. Jaundiced serum can also give abnormal results and are avoided as far as possible. Principle procedure for calculating results, including measurement uncertainty; Absorbance of Blank (AB), Sample Blank (ASB), Standard (AS) and Test (AT) measured against distilled water. AT - ASB Total ADA activity in U/L 	=		x 50 AS - AB Biological reference intervals; Less than 30 U/L is normal. 40 – 60 U/L – Suspected. >60 – Positive. Reportable interval of examination results. 2 – 5 days. Alert/critical values, where appropriate, Values above 50 U/L are alerted to the clinician immediately. Laboratory interpretation; Less than 30 U/L is normal. 40 – 60 U/L – Suspected. >60 – Positive. Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe disposal of waste materials including sample. Potential sources of variability. Lysed, jaundiced and lipemic samples can give abnormal values.

Estimation of Vanillyl Mandelic Acid (VMA) – Urine
Purpose of the examination In vitro quantitative measurement of vanillyl mandelic acid (VMA) concentration in patient’s urine. Principle of the procedure used for examinations; Solid phase ELISA based on competition between VMA coated on a microtiter well and that in urine for the monoclonal antibody. Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); All samples with VMA concentration greater than 8 µl/ml should be repeated at much higher dilutions (e.g., 1:20). Sensitivity is >0.0625 µg/ml. Minimal detectable concentration is 0.035 µg/ml. Primary sample collection (e.g. plasma, serum, urine); 24 hr urine sample is collected with 10 ml 6N HCl as preservative. Overnight or randomly collected urine should be acidified to a pH between 2 and 3 immediately after collection. Total volume is recorded and 1-5 ml urine is taken for analysis of VMA. All samples should be refrigerated until tested. Turbid samples should be centrifuged. Type of container and additives; 24 hr urine samples collected in 10 ml 6N HCl as preservative. Required equipment and reagents; ELISA plate reader, pH meter, 5N NaOH, 5N HCl, Saline solution, 0.01 M phosphate buffered saline (pH 7.4), pipette. Calibration procedures (metrological traceability); Method is calibrated and standardized at routine intervals to assess the reliability of the method. Procedural steps; 1.0 ml acidified urine is taken and transferred to a disposable tube in which pH of urine sample can be readjusted. pH of all samples is brought within the range of 6 – 9 by stepwise addition of small amounts of 5N NaOH while checking pH. pH readjusted sample is diluted 1:10 with phophate-buffered saline. The pH for diluted samples should range between 7.0–8.0. 50 µl VMA calibrators, controls and samples are dispensed into respective designated wells. 50 µl anti-VMA enzyme conjugate is dispensed into each well. Plate is mixed well and allowed to stand at 15 – 30 C for 1 hr. Incubation mixture is removed by decanting plate into a sink and blotting the paper on adsorbent paper. 300 µl normal saline is dispensed into each well. Saline is removed by decanting plate into a sink and blotting the paper on adsorbent paper. 100 µl VMA/HVA color developing reagent is dispensed to the well and allowed to stand at 15 – 30 C for 25’. 100 µl VMA/HVA stopping solution is added to the wells. Absorbance is measured at 450 nm. Quality control procedures; Method is calibrated and standardized at routine intervals to assess the reliability of the method. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Homovanillic acid, DL-3,4-Dihydroxymandelic acid, 3,4-Dihydroxy phenylacetic acid, Metanephrine, Vanillylpyruvic acid, Vanillic acid, Dopamine, 5 hydroxy 3 indole acetic acid, vanillyl lactic acidand 3 methoxy 4 hydroxy phenyl glycol can cause cross-reactivity. Principle procedure for calculating results, including measurement uncertainty; Calibration curve is generated by plotting VMA concentration s on X axis and absorbance on Y axis. VMA concentration for each unknown sample is obtained from the calibration curve. Final concentration (VMA mg.24 hrs) is obtained by the equation - VMA (µg/ml) x Urine volume (ml) / 1000. Biological reference intervals; Reference range for VMA / 24 hrs is < 15.0 mg/day. Reportable interval of examination results. 2 – 5 days. Alert/critical values, where appropriate, Abnormal values (anything > 10.0 mg/day) is alerted without delay to the concerned clinician. Laboratory interpretation; Reference range for VMA / 24 hrs is < 15.0 mg/day. Pheochromocytoma tumors can have values above that to very high levels. Safety precautions; Samples and reagents are handled with utmost care. Sample waste disposal is carried out using standard protocols. Potential sources of variability. Homovanillic acid, DL-3,4-Dihydroxymandelic acid, 3,4-Dihydroxy phenylacetic acid, Metanephrine, Vanillylpyruvic acid, Vanillic acid, Dopamine, 5 hydroxy 3 indole acetic acid, vanillyl lactic acidand 3 methoxy 4 hydroxy phenyl glycol can cause cross-reactivity.

Urine Porphyrin Screening
Purpose of the examination Semi-quantitative estimation and identification of Porphyrin in the given urine sample. Principle of the procedure used for examinations; With the exception of carboxy hemoglobin, quantitation of hemoglobin pigments is rarely necessary in clinical practice. Qualitative assessment is achieved by using spectroscopy. Wavelengths of absorption maxima for Porphyrin is measured at 407 nm. This is compared and quantitated against normal control and standard.

For the qualitative identification of porphobilinogen, Ehrlich’s aldehyde test is used. Porphobilinogens give a characteristic red color. Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); Semiquantitative screening test. Primary sample collection (e.g. plasma, serum, urine); Urine Type of container and additives; Plain clean container. No additives. Required equipment and reagents; Scanning spectrophotometer, Ehrlich’s reagent Calibration procedures (metrological traceability); The method is checked regularly to identify false positive and negative results with known standards. Procedural steps; With the exception of carboxy hemoglobin, quantitation of hemoglobin pigments is rarely necessary in clinical practice. Qualitative assessment is achieved by using spectroscopy. Wavelengths of absorption maxima for porphyrin is measured at 407 nm. This is compared and quantitated against normal control and standard. Qualitative estimation of porphobilinogen – Equal volumes of urine and Ehrlich’s reagent (0.7 gm p-dimethyl amino benzaldehyde in 150 ml HCl) are mixed and allowed to stand for 3’. Saturated aqueous solution of sodium acetate (2 volumes) added. After 3’, few ml of chloroform is added and shaken vigorously. Quality control procedures; Method is validated at regular intervals to check the validity of the technique. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Turbid urine should be filtered and used as it can give false results. Principle procedure for calculating results, including measurement uncertainty; Semi-quantitative assay, does not involve any major calculation, except spectrophotometric reading. Biological reference intervals; Semi-quantitatve method. Reported as either positive or negative. Optical density roughly corresponds to concentration of porphyrins in the sample. Reportable interval of examination results. 1 day. Alert/critical values, where appropriate, Any positive result is immediately informed. Laboratory interpretation; Porphobilinogen forms a red color which unlike that formed by uribilinogen is insoluble in chloroform. Any red color remaining in the aqueous phase after a second extraction with chloroform constitutes a positive test for porphobilinogen and is highly suggestive of acute porphyria.

Safety precautions; Sample and reagents are handled carefully. Disposal following all standard protocols. Potential sources of variability. Turbid urine can give false results. Qualitative Analysis of Mucopolysaccharides in Urine

Purpose of the examination Semi-quantitative determination of mucopolysaccharides in urine. For the diagnosis of mucopolysaccharidosis. Principle of the procedure used for examinations; Mucopolysaccharides give a blue color with Alcian blue stain, which is used for the identification of the condition. Intensity of color is roughly proportional to the concentration of mucolpolysacchrides in the sample. Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); Semi-quantitative assay. Primary sample collection (e.g. plasma, serum, urine); Urine, fresh and early morning samples are preferred. Type of container and additives; Plain clean container, no preservatives used. Required equipment and reagents; Whatmann No: 1 filter paper, Alcian blue stain. Calibration procedures (metrological traceability); Method is checked at regular intervals using known standards to assess reliability and reproducibility of results. Procedural steps; Spot urine 20 times on a Whatmann No: 1 filter paper. After spotting each time, allow the filter paper to dry. Stain the filter paper using Alcian Blue stain. Remove excess stain by washing with 5 % acetic acid. Allow the filter paper to dry and examine the color of spot.

Quality control procedures; Method is checked at regular intervals using known standards to assess reliability and reproducibility of results. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Turbid urine samples can give false results and hence have to be avoided. Principle procedure for calculating results, including measurement uncertainty; Mucopolysaccharides give blue color when stained with Alcian blue. False positive and negative do occur, but the rate of occurrence is less and this can be used as a reliable test for the easy detection of mucopolysaccharides in urine. Biological reference intervals; Semiquantitative assay. Normally urine will not contain mucopolysaccharides. Will not give any spot. Intensity of spot corresponds to concentration of mucopolysaccharide present. Reportable interval of examination results. 1 day. Alert/critical values, where appropriate, Any sample positive for mucopolysaccharide is immediately informed to the treating clinician. Laboratory interpretation; Blue spot with Alcian blue is positive. Intensity of blue color is roughly proportional to the concentration of mucopolysaccharides in urine. Safety precautions; Sample and reagents are handled very carefully. Sample disposal is done following all safety precautions. Potential sources of variability. False positive and negative results may occur at a frequency of roughly 1 %.

Screening Test for Homocysteine in Urine
Purpose of the examination Qualitative determination of homocysteine in urine. For the diagnosis of homocysteinuria. Principle of the procedure used for examinations; Homocysteine gives a purple colored ring along with silver nitrate/sodium cyanide which is detected by this useful screening test. Other sulphur containing amino acids can also give a false positive result. Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); Qualitative test. These parameters do not apply. Primary sample collection (e.g. plasma, serum, urine); 5 ml Urine Type of container and additives; Plain clean container Required equipment and reagents; Sodium chloride, ammonia, silver nitrate, sodium nitroprusside, sodium cyanide. Calibration procedures (metrological traceability); Method is checked at regular intervals to check the validity and the stability of reagents using known standards. Procedural steps; To 1 ml of deproteinized, desalted urine sample, NaCl is added to saturation. Then, 0.2 ml silver nitrate (in ammonia) and 0.2 ml sodium cyanide (in water) are added. This is followed by 0.2 ml freshly prepared sodium nitroprusside (in water) along the sides of the test tube. Quality control procedures; Method is checked at regular intervals to check the validity and the stability of reagents using known standards. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Other sulphur containing amino acids (Cysteine, cystine, homocystine) can also give pinkish purple ring. Principle procedure for calculating results, including measurement uncertainty; A pinkish purple ring appearing within 2 min after the addition of sodium nitroprusside is positive for Homocysteine. Biological reference intervals; Qualitative test. Not applicable. Reportable interval of examination results. 1 day. Alert/critical values, where appropriate, All positive results are immediately informed to the treating clinician. Laboratory interpretation; A pinkish purple ring appearing within 2 min after the addition of sodium nitroprusside is positive for Homocysteine Safety precautions; Cyanide is a source of extreme caution in this test. It is handled and stored with extreme care. Only experienced technicians are allowed to do this test, under strict supervision. Potential sources of variability. Other sulphur containing amino acids (Cysteine, cystine, homocystine) can also give pinkish purple ring.

Estimation of 17 Hydroxy Progesterone
Purpose of the examination Enzyme immunoassay for the determination of 17 hydroxy progesterone in human serum and plasma.

Principle of the procedure used for examinations; Solid phase ELISA based on competitive assay principle. Unknown quantity of antigen in sample and fixed amount of enzyme labeled antigen compete for binding sites on antibody coated into wells of the ELISA plate. After substrate reaction, intensity of color developed is inversely proportional to antigen concentration. Standard curve is plotted and results can be read from it.

Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity);

Linearity range is 1.56 – 9.23 ng/ml. Analytical sensitivity is 0.03 ng/ml. Precision is 2.44 – 11.41 ng/ml (Intra assay) and 0.26 – 5.74 ng/ml (Inter assay).

Primary sample collection (e.g. plasma, serum, urine); Serum / Plasma (EDTA is used as anti-coagulant).

Type of container and additives; EDTA anti-coagulant. Required equipment and reagents; ELISA reader, microtiter plate, enzyme conjugate, Standards, Reagents (supplied by manufacturer), Orbital shaker, micropipettes, vortex mixer. Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results. Procedural steps; 1.	Pipette 25 µl of each standard, Control and sample into respective wells of microtiter plates. 2.	Pipette 200 µl of enzyme conjugate into each well. 3.	Cover plate with adhesive foil. Shake plate carefully. Incubate 60 min at RT (18-25 C). 4.	Remove adhesive foil. Discard incubation solution. Wash plate 3 x with 250 µl of diluted wash buffer. Remove excess solution by tapping the inverted plate on a paper towel. 5.	100 µl of TMB substrate solution added into each well. 6.	Incubate 30 min at RT (18-25 C). 7.	Stop the substrate reaction by adding 100 µl of TMB stop solution into each well. Briefly mix contents by gently shaking the plates. 8.	Optical density measured at 450 nm within 30 min after pipetting the stop solution. Quality control procedures; Internal Quality Control – Multiple standards (7 of different concentrations) run along with each batch of analysis. Standard curves plotted and validated with each batch. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Lipaemia, hemolysis and bilirubinemia to be avoided, as all these can cause erroneous results. Cross – reactivity can occur with 17  OH pregnenolone, progesterone, 11 deoxy cortisol, desoxy corticosterone, cortisol and pregnenolone. Principle procedure for calculating results, including measurement uncertainty;

Obtained optical density is plotted against concentration using automated method. Concentration of samples directly read from the standard curve. Samples showing signals above the highest standard are diluted and reassayed.

Biological reference intervals;

Healthy individuals have 17 OH progesterone in the range 0.05 – 1.6 ng/ml. Values are slightly higher in the luteal phase (0.2 – 2.9 ng/ml) than in the follicular phase (0.3 – 1.0 ng/ml). In pregnancy values are higher, 0.4 – 15.4 ng/ml. Reportable interval of examination results. 2-5 days. Alert/critical values, where appropriate, Values above 10.0 ng/ml are alerted to the clinician immediately. Laboratory interpretation; Healthy individuals have 17 OH progesterone in the range 0.05 – 1.6 ng/ml. Values are slightly higher in the luteal phase (0.2 – 2.9 ng/ml) than in the follicular phase (0.3 – 1.0 ng/ml). In pregnancy values are higher, 0.4 – 15.4 ng/ml. Safety precautions; Sample and reagents are handled carefully. Special precaution is given for disposal of sample as well. Potential sources of variability. Lipaemia, hemolysis and bilirubinemia are to be avoided.

Lipoprotein Electrophoreis for Plasma Lipoproteins
Purpose of the examination Lipoprotein electrophoresis procedure is intended for separation and quantitation of plasma lipoproteins by cellulose acetate electrophoresis. Principle of the procedure used for examinations; The specimen is applied to an agarose plate that has been presoaked in a tris-barbital buffer at pH 8.8. The lipoprotein fractions are separated by electrophoresis and then stained with a methanol solution of Sudan Black at an alkaline pH. The stained bands may be visually inspected for qualitative results or may be quantitated in scanning densitometer using a 525 nm filter. Primary sample collection (e.g. plasma, serum, urine); Plasma Type of container and additives; EDTA plasma Required equipment and reagents; Electrophoresis chamber, Barbital buffer, Sudan Black stain, Agarose plates Calibration procedures (metrological traceability); Method is checked at regular intervals using known samples from known patients to assess the reliability of the method. Procedural steps; 1.	Sudan Black dye is made in ethylene glycol (5 g/L) in ethylene glycol. 2.	Barbitone buffer – Is made in lukewarm water at pH 8.6. Buffer is always used at room temperature. 3.	Sample preparation – Sample is mixed with dye. 200 µl serum and 300 µl dye. Kept at room temperature for 4 hrs. Sample is activated by placing vials at 42 C for 10’. 4.	Slides – Clean slides with alcohol, weigh agarose (1%) and heat to boil. Pour hot agarose on slides, exactly 2-5 ml fills one slide. Allow to set and cut wells. Mark control and test separately, on separate slides. 5.	Electrophoresis – Pour buffer into tanks, make connections and make wicks. Add 20-30 µL dye/sample mixture wells. Place the slides and wicks and run at 150 V for 35 – 45 min. Observe the bands. 6.	Buffer can be re-used after run. Quality control procedures; Method is checked at regular intervals using known samples from known patients to assess the reliability of the method. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Heparin therapy causes activation of lipoprotein lipase, which increases the relative migration rates of the fractions, especially the beta lipoprotein. Principle procedure for calculating results, including measurement uncertainty; Principally involves visualizing the slide and reading under a densitometric scanner. Biological reference intervals; Normal Range: Alpha: 23.1-54.5 % Pre-Beta: 10.1-35.9 % Beta: 30.6-53.9 % Chylomicrons 0- 1.9 % Reportable interval of examination results. 1-2 days. Alert/critical values, where appropriate, Any abnormal pattern detected is immediately informed to clinicians. Laboratory interpretation; Normal Range: Alpha: 23.1-54.5 % Pre-Beta: 10.1-35.9 % Beta: 30.6-53.9 % Chylomicrons 0- 1.9 % Gel is also interpreted visually for bands and correlated with lipid profile parameters. Advantage for lipoprotein electrophoresis is the detection of chylomicons that is not possible by routine lipid profile studies. Safety precautions; All samples and reagents are handled with utmost care. Samples are disposed following standard disposal protocols. Potential sources of variability. Diet can influence the pattern to some degree. High fat diet can have elevated levels of chylomicrons. Hence fasting sample is best used for the assay.

Estimation of Vitamin B12 in Serum
Purpose of the examination For the in vitro quantitative measurement of vitamin B12 in human serum and plasma (EDTA or heparin), to aid in the differential diagnosis of anemia. Principle of the procedure used for examinations; The Vitros Immunodiagnostic Products Vitamin B12 assay is performed using the Vitros Vitamin B12 Reagent Packs 1 and 2, the Vitros Immunodiagnostic Products Vitamin B12/Folate Reagent Pack 3 and the Vitros Immunodiagnostic Products Vitamin B12 Calibrators on the Vitros Immunodiagnostic System. A competitive binding technique is used. This depends on competition between vitamin B12 present in the sample and horseradish peroxidase (HRP)-labeled vitamin B12 for a limited number of binding sites on a biotinylated Intrinsic Factor present in the liquid phase. Vitamin B12 present in patient samples is released from its endogenous binding proteins by alkaline denaturation. Biotinylated Intrinsic Factor conjugate is added and incubated with the treated neutralized sample. An aliquot of the treated sample is transferred into a streptavidin coated well and B12-HRP conjugate added. Following a competitive binding reaction, the vitamin B12 Intrinsic Factor complex is captured by streptavidin on the well. Unbound conjugate is removed by washing. The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the Vitros System. The amount of HRP conjugate bound is inversely proportional to the concentration of vitamin B12 present. Primary sample collection (e.g. plasma, serum, urine); Serum / Plasma Type of container and additives; Serum, 5 ml Required equipment and reagents; Reagents provided by manufacturer, wash reagents Calibration procedures (metrological traceability); Method is calibrated and validated at regular intervals using known standards and calibrators. Procedural steps; The Vitros Vitamin B12 assay requires 30 µL sample, calibrator or control for a singleton determination. This does not take account of the minimum fill volume of the chosen sample container. The Vitros Vitamin B12 assay must be calibrated each time a new reagent lot (Reagent Pack 1) is used, and subsequently at intervals of 28 days. The Vitros Vitamin B12 assay may also need to be calibrated after certain service procedures are performed or if quality control results are consistently outside your acceptable range. 1.	Scan the protocol card to load a new assay protocol onto the System. The assay button is then displayed on the Sample Programming screen. Scan the lot calibration card for each new lot of Vitamin B12 Reagent Pack 1, Reagent Pack 2 and Vitamin B12/Folate Reagent Pack 3 to enter lot specific calibration (Reagent Pack 1) and expiration information. 2.	Open the foil pouches and remove the reagent packs. Load the packs at the autoload station, or use the Unload/Load button on the Reagent Management - View by Reagent screen. Ensure that Reagent Packs 1, 2 and 3 have been loaded and sufficient reagents are available in each pack for the work planned. The names that will appear on the Reagent Management Screen are B12 (Reagent Pack 1), P2B12 (Reagent Pack 2) and P3 BF (Reagent Pack 3). Note: Do not use damaged or incompletely sealed product. 3.	Load samples into universal sample trays, using adapters where necessary (samples may be bar coded if desired). Place a disposable tip adjacent to each sample and load the trays onto the System. Define sample programs using the Sample Programming screen. Ensure that sufficient disposable tips are available in the auxiliary tip supply for the work planned. Start the sampling operation, all sample processing steps will be carried out automatically. 4.	Process calibrators in the same manner as samples. Calibration need not be programmed if bar code labels are used; load the calibrators in any order, calibration will be initiated automatically. Quality control procedures; Method is calibrated and validated routinely using known calibrators and standards to assess the quality of the method. Interferences (e.g., lipaemia, haemolysis, bilirubinemia) and cross reactions; Samples containing triolein, hemoglobin or bilirubin interfere with this assay by less than 10%. Do not use turbid samples. Biotin levels in serum remain elevated for up to 24 hours after oral or intravenous biotin administration. Samples containing 20 ng biotin/mL interfere by less than 10%. Cobinamide dicyanide can cause cross- reactivity to the extent of 0.002 %. Biological reference intervals; 200 – 900 pg/ ml Reportable interval of examination results. 5 – 7 days. Alert/critical values, where appropriate, Abnormal values (<150 pg/ml and >1000 pg/ml) are alerted to the concerned clinician. Laboratory interpretation; Values less than 100 pg/ ml is signs of Vitamin B12 deficiency. Higher values above 1000 pg/ml are indicative of Vitamin B12 excess, is not as significant as deficiency. Safety precautions; All samples and reagents are handled carefully. Waste disposal is carried out fllowing standard protocols. Potential sources of variability. Samples containing triolein, hemoglobin or bilirubin interfere with this assay by less than 10%. Do not use turbid samples. Biotin levels in serum remain elevated for up to 24 hours after oral or intravenous biotin administration. Samples containing 20 ng biotin/mL interfere by less than 10%. Cobinamide dicyanide can cause cross- reactivity to the extent of 0.002 %.

Principle of measurement IN CBC ANALYSER:
WBC Analysis WBCs are analyzed in two separate channels optical (WOC) and impedance (WIC). WOC measurement: The WOC sheath syringe dispenses 1.6 ml of sheath reagent through the shear valve. The sample segment and sheath are then routed to the WOC mixing chamber, where the dilution is bubble mixed. The final; dilution is 1:51. The stream of WOC sheath reagent is directed through the flow cell. The dilution is hydrodyanamically focused into a narrow stream. A laser beam is focused on the flow cell. As the sample stream intersects the laser beam, the light scattered by the cells is measured at four different angular intervals. WIC measurement: The WIC / HGB diluent syringe dispenses 5.25ml of diluent through the shear valve, where it picks up 20ul sample. The dilution is then bubble mixed. (1:301 dilution). The dilution is pulled through the aperture by vacuum, process known as volumetric metering. Electrical impedance is used to count the WBCs as they traverse the hole. RBC / PLTanalysis: The RBC diluent syringe dispenses 8.0 ml of diluent through the shear valve, where it takes0.74ul sample. The mixture is bubble mixed in the RBC / PLT transducer (1:10,812)\ The dilution is pulled through the aperture by vaccum, volumetric metering. Electrical impedance is used to count the RBCs and PLTs. Haemoglobin analysis: After 200ul of the WIC / HGB dilution are metered through the WIC aperture,the remaining dilution is transferred to the HGB flow cell. The HGB concentration is measured spectrophotometrically.

== Reticulocyte count

Principle for manual method. ==

The remnants of ribosomal ribonucleic acid have the property of binding certain basic dyes brilliant cresyl blue or new methylene blue to form a blue or purple precipitate of filaments. This reaction takes place only in vitally stained unfixed preparations. Performance specification Microscopic examination Sample EDTA anticoagulated blood Type of container EDTA tube Equipments and reagents 1)	1%Brilliant cresyl blue Brilliant cresylblue	-1gm Sodium chloride	-0.7gm Sodium citrate	-0.6gm Distilled water	-100ml Store in dark bottle under refrigeration, filter before use. 2) EDTA blood 3) 370c water bath. 4) Clean glass slides 5) Microscope

Procedure Deliver 2 to 4 drops of the dye solution in to a 75x10mm glass or plastic tube. 	Add 2-4 drops of the patients EDTA anticoagulated blood to the dye solution and mix, keep the mixture at 370c for 15-20 min 	Resuspended the red cells by gentle mixing and make smears on glass slides 	When dry, examine the smear under oil immersion objective 	Count systematically at least 500RBCs including in this the number of reticulocytes Calculation No:of reticulocytes in ‘n’ field		= X Average no:of red cells per field		= y Total no: of red cells in’n’field		= n x y Recticulocyte percentage 		        = [X/n x y] x 100% Absolute reticulocyte count		        = Retic % x RBC

Reticulocyte Percentage Average reticulocyte per oil immersion x 100 Average total RBCs per oil immersion

For Example:

If 400,700,450,600,350 RBCs are seen in five fields. Then the average RBC count = 400+700+450+600+350=500 5 If 5,2,3,1,0 reticulocytes are seen in five different fields. Then average reticulocyte count = 5+2+3+1+0= 3 Reticulocyte Percentage = 3/500 x 100=0.6% Normal range Adult       		 - 0.2 – 2% Children’s 		 - 0.5 - 4% Infants     		 - 2 – 6%

RETICULOCYTE PRODUCTION INDEX % of Retic x Pt’s HCT x     1 RPI =	0.45           correction factor {0.45 = normal HCT} Correction factor

Pt’s HCT value in %	Correction factor

40-45	1.0 35-39		1.5 25-34		2.0 15-24		2.5 < 15	3.0

Corrected reticulocyte count		= % of observed retic x Pt’s HCT

0.45 RPI less than 2 	    failure of BM red all production or a hypoproliferative anaemia RPI of 3 or more 	     Marrow hyper proliferation or an appropriate response. Example % Retic = 3% then RPI  = 3 x 42 x  1 0.45	1 =0.567 [Correction factor: - 1.0] Normal range Reticulocyte count In adults:	0.5-2% In infants:  2-6% Interpretation Reticulocyte count are low in ineffective erythropoiesis,: eg myelosclerosis, aplastic anaemia, megaloblastic anaemia, thalassaemia, and sideroblatic anaemia   Reticulocytosis occur after blood loss or effective therapy for certain kinds of anaemia Eg: therapy of iron deficiency anaemia, megaloblastic anaemia and also occur in haemolytic anaemias. Safety precautions: Refer Laboratory Safety Manual Reference : 1.Operator’s manual CELL-DYN 3500 by Abbott diagnostics. 2.Basic haematological techniques, Dacie and Lewis Practical haematology, 9th edition; 2001,3;29.

SEDATIVES
1.1 Purpose of the examination To screen for the presence of Sedatives 1.	Common Barbiturates 2.	 Benzodiazepines 3.	Chloralhydrate

1.2 Principle of the procedure used for examinations; Preliminary qualitative test using Test Cassette followed by thin         layer chromatography is performed on Gastric aspirate, urine and blood samples.

1.2.1 Barbiturates-Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy-colored conjugate pad containing mouse anti-barbiturate antibodies coupled to colloidal gold; and 2) nitrocellulose membrane containing a Test (T) line and a Control (C) line. The test line is coated with barbiturate-BTG, and the Control line is coated with goat –anti -rabbit IgG antibody.

This test is a competitive binding immunoassay. The barbiturate in the urine specimen competes with the barbiturate-BTG antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated anti-barbiturate antibodies.

When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of barbiturate in the urine specimen is below the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of barbiturate in the urine specimen is at or above the cutoff, no Test line develops. The C line binds to the gold-conjugated rabbit IgG forms a burgundy color line, regardless of the presence of barbiturate.

1.2.2 Benzodiazepines-Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy –colored conjugate pad containing mouse antioxazepam antibodies coupled to colloidal gold, and 2) nitrocellulose membrane containing a Test (T) line and a Control (C) line. The Test line is coated with benzodiazepines –BSA, and the Control line is coated with goat anti-mouse IgG antibody.

This test is a competitive binding immunoassay. The oxazepam in the urine specimen competes with the benzodiazepines-BSA antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated antigen oxazepam antibodies.

When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of oxazepam in the urine specimen is at or above the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of oxazepam in the urine specimen is at or above the cutoff, no Test line develops.

The control line is coated with goat anti-mouse antibody, which should bind to the gold-antibody conjugate and form a burgundy color line regardless of the presence of oxazepam.

1.2.3 Benzodiazepines and Barbiturates - TLC Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer.

For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualisation of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

1.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

1.4  Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50ml) Gastric aspirate (10-25 ml)

1.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

1.6  Required Equipment and Reagents; Screening is done and interpretation is made manually. No  equipments are needed for this purpose. Thin layer chromatography      is done in a Chromatography chamber.

1.7 Calibration Procedures (Metrological Traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

1.8  Procedural Steps; 1.8.1 Chromatographic Immunoassay 1.8.1.1 Barbiturates 1.8.1.1.1  Precaution •	The instructions must be followed exactly to obtain accurate results •	Do not open the sealed pouch, unless ready to conduct the assay •	Do not use expired devices •	Dispose of all specimens and used assay materials as potentially biohazardous.

1.8.1.1.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibriated to room temperature before testing. •	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding dropper vertically, add four drops of the specimens to the sample well. •	Read the test result between four (4) to seven (7) minutes after adding the specimen.

1.8.1.1.3 Interpretation of results. Important: Do not read test results after seven (7) minutes. The T line should be interpreted independently of the C line.

C	      C	             C	  C              T	       T	             T	  T                  (+)                (-) Positive       Negative               Invalid

Positive: If only the C line appears, the test indicates that the oxazepam level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative: If both C line and T line appear, the test indicates that the oxazepam level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

1.8.1.2 Benzodiazepines 1.8.1.2.1 Precaution •	The instructions must be followed to obtain accurate results. •	Do not open the sealed pouch, unless ready to conduct the assay. •	Do not use expired devices. •	Dispose of all specimens and used assay materials as potentially biohazards. •	1.8.1.2.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibriated to room temperature before testing. •	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding the dropper vertically, add four drops of the specimen to the sample well marked as”S”on the device. Note: If migration is not observed in 30 seconds in the results window, add one drop of urine specimen Read the test result between four (4) to seven (7) minutes after adding the specimen. Important: Do not read test results after seven (7) minutes.

1.8.1.2.3 Interpretation of results:

C	      C	            C	  C             T	       T	            T	  T                  (+)                (-) Positive       Negative              Invalid Positive: If only the C line appears, the test indicates that the oxazepam level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative: If both C line and T line appear, the test indicates that the oxazepam level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

1.8.2  Thin Layer Chromatography AIM – Separation and identification of the Barbiturate and Benzodiazepine compound present in the sample

1.8.2.1 Reagents and materials Standard Barbiturate and Benzodiazepine compound, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and samples.

1.8.2.2	Method 1.8.2.2.1 Extraction procedure for Barbiturates and       Benzodiazepines from urine and gastric aspirate. Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

1.8.2.2.2 Extraction procedure for Barbiturates and       Benzodiazepines  from  blood sample. Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allowed to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drained out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

1.8.2.2.3 Sample Application on TLC Plate and Development of Chromatogram Reconstitute the extract with methanol. Tank is saturated with the solvent for a minimum of 30 minutes. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness) Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed in the tank for run to occur; taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

1.8.2.3 Result Standard Barbiturate and Benzodiazepine compounds gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Barbiturate and Benzodiazepine compounds are present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

1.8.2.2.4 Chloral hydrate: Reagents: Aqueous sodium hydroxide solution (5 mol/l, i.e; 200 g/l). Aqueous trichloroacetic acid (10 mg/l).

Method To separate 10-ml tubes add 1-ml portions of: (a)	Test urine; (b)	Purified water; and (c)	Trichloracetic acid solution. (d)	Add 1 ml of sodium hydroxide solution and 1 ml of pyridine to each tube, mix gently and fit with a loose stopper. (e)	Heat in a boiling water-bath for 2 minutes.

Results as intense red/purple colour in the upper, pyridine layer indicates the presence of trichloro compounds. The blank analysis excludes contamiation with compounds such as chloroform from the laboratory atmosphere. Compounds such as chloral hydrate, dichloralphenazone and trichloroethylene, which are extensively metabolized to trichloroacetic acid; give strong positive results in this test.

1.8.2.4 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

1.9  Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

1.10	Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 1.10.1 For Chromatographic Immuno assay Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

1.10.2	For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required. 1.11Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative.

1.12.  Reportable interval of examination results. Same day

1.13  Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

1.14  Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

1.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe disposal of waste materials including sample.

1.16 References 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation.

3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical      Press, London, 2004.

Analgesics and Anti-inflammatories
2.1 Purpose of the examination To screen for the presence of Analgesics and Anti- inflammatories 1.	Salicylates 2.	Paracetamol 3.	Selected NSAIDs

2.2 Principle of the procedure used for examinations; Colour tests followed by thin layer chromatography are performed on gastric aspirate, urine and blood samples.

2.2.1 Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer. For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin 2.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

2.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50ml) Gastric aspirate (10-25 ml)

2.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

2.6 Required equipment and reagents Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

2.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

a.	Procedural steps; 2.8.1 Salicylates  Qualitative test Applicable to urine, stomach contents and scene residues.

2.8.1.1 Reagents Trinder’s reagent. Mix 40 g of mercuric chloride dissolved in 850 ml of purified water with 120ml of aqueous hydrochloric acid ( colour indicates the presence of salicylates. This test is sensitive and will detect therapeutic dosage with salicylic acid, acetylsalicylic acid, 4-aminosalicylic acid, methyl salicylate and salicylamide.

2.8.1.4 Sensitivity Salicylate, 10mg/l.

2.8.2 Paracetamol  Qualitative test Applicable to urine, stomach contents and scene residues.

2.8.2.1 Reagents 1.Concentrated hydrochloric acid (relative density 1.18). 2.Aqueous 0-cresol solution (10g/l). 3.Aqueous ammonium hydroxide solution (4 mol/l).

2.8.2.2 Method •	Add 0.5 ml of hydrochloric acid to 0.5 ml of sample, boil for 10 minutes and cool. •	Add 1 ml of o-cresol solution to 0.2 ml of the hydrolysate. •	Add 2 ml of ammonium hydroxide solution and mix for 5 seconds.

2.8.2.3 Results A strong, royal blue colour developing immediately indicates the presence of paracetamol. This test is very sensitive and will detect therapeutic dosage with paracetamol 24-48 hours later.

2.8.2.4 Sensitivity p-Aminophenol, 1 mg/L.

2.8.3 Thin Layer Chromatography Aim – Separation and identification of the Analgesics and Anti-inflammatory compounds present in the sample

2.8.3.1Reagents and Materials Standard Analgesics and Anti-inflammatory compounds, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

2.8.3.2 Method 2.8.3.2.1 Extraction procedure for Analgesics and Anti Inflammatories from urine and gastric aspirate. Take10 ml of sample in separating funnel; add few drops tartaric  acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness. 2.8.3.2.2Extraction procedure for Analgesics and Anti Inflammatories from blood sample. Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

2.8.3.2.3 Sample Application on TLC plate and development of chromatogram. Tank is saturated with the solvent for a minimum of 30 minutes. Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness) Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed in the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

2.8.3.3 Result Standard Analgesics and Anti-inflammatory compounds gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Analgesics and Anti-inflammatory compounds are present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

2.8.3.4 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

2.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

2.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia)          and cross reactions; 2.10.1 Trinders Test Azide preservatives react strongly in this test, and weak false positives can be given by urine specimens containing high concentrations of ketones (ketone bodies).

2.10 .2 O-Cresol test Only aromatic amines, such as aniline, which also give rise to                          p-aminophenol in urine after hydrolysis are known to interfere.

2.10.3 For Thin Layer Chromatography Chromatography of compounds that originate from biological          extracts shows interferences from additional material present in    sample extracts (matrix effects). For eg extracts of stomach contents   may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

2.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative. 2.12  Reportable interval of examination results. Same day 2.13  Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

2.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretation is made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

2.15   Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe disposal of waste materials including sample.

2.16 	References; 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation. 3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004.

Phenothiazines 3.1  Purpose of the examination To screen for the presence of Phenothiazines

3.2  Principle of the procedure used for examinations; Colour tests followed by thin layer chromatography is performed on gastric aspirate, urine and blood samples.

3.2.1 Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer.

For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

3.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this sections do not apply.

3.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

3.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

3.6 Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

3.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

3.8 Procedural steps; 3.8.1 Qualitative test Applicable to urine, stomach contents and scene residues.

3.8.1.1Reagents FPN reagent. Mix 5 ml of aqueous ferric chloride solution (50g/l), 45ml of aqueous perchloric acid (200g/kg) and 50 ml of aqueous nitric acid (500ml/l)

3.8.1.2 Method Add 1 ml of FPN reagent to 1 ml of sample and mix for 5 seconds.

3.8.1.3 Results Colours ranging from pink, red or orange to violet or blue may indicate the presence of phenothiazines or metabolites. Urine from patients on chronic treatment with conventional phenothiazines, such as chlorpromazine, will usually give a positive reaction. 3.8.1.4 Sensitivity Chlorpromazine, 25 mg/l.

3.8.2 Thin Layer Chromatography Aim – Separation and identification of the Phenothiazine compound present in the sample. 3.8.2.1 Reagents and Materials Standard Phenothiazine compound, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

3.8.2.2 Method 3.8.2.2.1Extraction procedure for Phenothiazines from urine and gastric aspirate: Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

3.8.2.2.2 Extraction procedure for Phenothiazines From blood sample: Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

3.8.2.2.3 Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness) Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

3.8.2.3 Result Standard Phenothiazine compounds gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Phenothiazine compounds are present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

3.8.2.4 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

3.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

3.10	Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 3.10.1 For Qualitative test Tricyclic antidepressants such as Imipramine may also give green or blue colours. False reactions may be obtained in patients with phenylketonuria or hepatic impairment.

3.10.2	For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

3.11Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative. 3.12 Reportable interval of examination results. Same day 3.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

3.14  Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

3.15	Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe               disposal of waste materials including sample.

3.16	Reference 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation.

3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004. Barbiturates 4.1 Purpose of the examination To screen for the presence of Barbiturates

4.2 Principle of the procedure used for examinations; Preliminary qualitative test using Barbiturate test cassette followed by thin layer chromatography is performed on gastric aspirate, urine and blood samples.

4.2.1	Barbiturates-Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy-colored conjugate pad containing mouse anti-barbiturate antibodies coupled to colloidal gold; and 2) nitrocellulose membrane containing a Test (T) line and a Control (C) line. The test line is coated with barbiturate-BTG, and the Control line is coated with goat –anti -rabbit IgG antibody.

This test is a competitive binding immunoassay. The barbiturate in the urine specimen competes with the barbiturate-BTG antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated anti-barbiturate antibodies.

When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test

strip. If the level of barbiturate in the urine specimen is below the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of barbiturate in the urine specimen is at or above the cutoff, no Test line develops. The C line binds to the gold-conjugated rabbit IgG forms a burgundy color line, regardless of the presence of barbiturate.

4.2.2	Barbiturates- Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer.

For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

4.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this sections do not apply.

4.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

4.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

4.6 Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

4.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

4.8 Procedural Steps; 4.8.1 Chromatographic Immunoassay Barbiturates 4.8.1.1 Precaution •	The instructions must be followed exactly to obtain accurate results •	Do not open the sealed pouch, unless ready to conduct the assay •	Do not use expired devices •	Dispose of all specimens and used assay materials as potentially biohazardous. •	4.8.1.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibrated to room temperature before testing. •	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding dropper vertically, add four drops of the specimens to the sample well •	Read the test result between four (4) to seven (7) minutes after adding the specimen.

4.8.1.3 Interpretation of results. Important: Do not read test results after seven (7) minutes. The T line should be interpreted independently of the C line.

C	      C	            C	  C               T	       T	            T	  T                  (+)                (-) Positive        Negative               Invalid

Positive: If only the C line appears, the test indicates that the barbiturates level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative If both C line and T line appear, the test indicates that the barbiturate level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

4.8.2 Thin Layer Chromatography Aim– Separation and identification of the Barbiturate compound present in the sample.

4.8.2.1 Reagents and Materials Standard Barbiturate compound, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

4.8.2.2 Method 4.8.2.2.1 Extraction procedure for Barbiturates from urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

4.8.2.2.2 Extraction From blood sample. Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

4.8.2.2.3 Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes. Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness) Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

4.8.2.2.4 Result Standard Barbiturate compounds gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Barbiturate compounds are present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

4.8.2.2.5 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

4.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

4.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 4.10.1   For Chromatographic Immuno assay Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

4.10.2	 For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

4.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative.

4.12 Reportable interval of examination results. Same day

4.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

4.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

4.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe disposal of waste materials including sample.

4.16 Reference: 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation. 3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004. Benzodiazepines 5.1 Purpose of the examination To screen for the presence of Benzodiazepines

5.2 Principle of the procedure used for examinations; Screening test using Benzodiazepine test cassette, followed by thin layer chromatography is performed on gastric aspirate, urine and blood samples.

5.2.1 Benzodiazepines-Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy –colored conjugate pad containing mouse antioxazepam antibodies coupled to colloidal gold, and 2) nitrocellulose membrane containing a Test (T) line and a  Control (C) line. The Test line is coated with benzodiazepines –BSA, and the Control line is coated with goat anti-mouse IgG antibody.

This test is a competitive binding immunoassay. The oxazepam in the urine specimen competes with the benzodiazepine BSA      coated on the nitrocellulose membrane for the limited binding sites of the conjugated antigen oxazepam antibodies.

When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of oxazepam in the urine specimen is at or above the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of oxazepam in the urine specimen is at or above the cutoff, no Test line develops.

The control line is coated with goat anti-mouse antibody, which should bind to the gold-antibody conjugate and form a burgundy color line regardless of the presence of oxazepam

5.2.2	Benzodiazepine –Thin Layer Chromatography. Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer.

For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

5.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

5.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

5.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

5.6 Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

5.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

5.8 Procedural steps; 5.8.1 Benzodiazepines  chromatographic Immuno Assay

5.8.1.1 Precaution •	The instructions must be followed to obtain accurate results. •	Do not open the sealed pouch, unless ready to conduct the assay. •	Do not use expired devices. •	Dispose of all specimens and used assay materials as potentially biohazards.

5.8.1.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibrated to room temperature before testing. •	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding the dropper vertically, add four drops of the specimen to the sample well marked as”S”on the device. •	Note: If migration is not observed in 30 seconds in the results window. Add one drop of urine specimen •	Read the test result between four (4) to seven (7) minutes after adding the specimen. •	Important: Do not read test results after seven (7) minutes.

5.8.1.3 Interpretation of results:

C	      C	            C	  C              T	       T	            T	  T                  (+)                (-) Positive        Negative              Invalid

Positive: If only the C line appears, the test indicates that the oxazepam level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative: If both C line and T line appear, the test indicates that the oxazepam level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device

5.8.2	Thin Layer Chromatography AIM – Separation and identification of the Benzodiazepine compound present in the sample.

5.8.2.1	Reagents and materials Standard Benzodiazepine compound, Solvent (Chloroform: Acetone, (4:1) & Methanol: Ammonia, (100:1.5), prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

5.8.2.2.Method 5.8.2.2.1 Extraction procedure Benzodiazepines From urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

5.8.2.2.2  Extraction procedure for Benzodiazepines From blood sample: Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

5.8.2.2.3	Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes Reconstitute the extract with methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness). Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front.

Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

5.8.2.2.4 Result Standard Benzodiazepine compounds gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Benzodiazepine compounds are present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

5.8.2.2.5 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

5.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

5.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 5.10.1   For Chromatographic Immuno assay

Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

5.10.2	For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

5.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative.

5.12 Reportable interval of examination results. Same day

5.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

5.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

5.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe disposal of waste materials including sample. Amitriptyline 6.1 Purpose of the examination To screen for the presence of Amitriptyline

6.2 Principle of the procedure used for examinations; Preliminary qualitative test using TCA test cassette, followed by thin layer chromatography is performed on gastric aspirate, urine and blood samples.

6. 2.1 Amitriptyline -Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy-colored conjugate pad containing mouse anti- Amitriptyline antibodies coupled to colloidal gold; and 2) nitrocellulose membrane containing a Test (T) line and a Control (C) line. The test line is coated with Amitriptyline-BSA, and the Control line is coated with goat –anti -rabbit IgG antibody. This test is a competitive binding immunoassay. The Amitriptyline in the urine specimen competes with the Amitriptyline -BSA antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated anti-amitriptylene antibodies.

When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of Amitriptyline in the urine specimen is below the cutoff (1000ng/ml), the Test line should appear as a visible burgundy line. If the level of Amitriptyline in the urine specimen is at or above the cutoff, no Test line develops. The control line is coated with goat anti-mouse antibody, which should bind to the gold-antibody conjugate and form a burgundy color line regardless of the presence of Amitriptyline.

6.2.2	Amitriptyline-Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer.

For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

6.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

6.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

6.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

6.6 Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

6.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

6.8 Procedural steps; 6.8.1 Chromatographic immuno assay for Amitriptyline 6.8.1.1 Precaution •	The instructions must be followed exactly to obtain accurate results •	Do not open the sealed pouch, unless ready to conduct the assay •	Do not use expired devices •	Dispose of all specimens and used assay materials as potentially biohazardous. •	6.8.1.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibrated to room temperature before testing. •	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding dropper vertically, add four drops of the specimens to the sample well •	Read the test result between four (4) to seven (7) minutes after adding the specimen. •	6.8.1.3 Interpretation of results. Important: Do not read test results after seven (7) minutes. The T line should be interpreted independently of the C line.

C	      C	            C	  C            T	       T	            T	  T                 (+)                 (-) Positive       Negative              Invalid Positive: If only the C line appears, the test indicates that the Amitriptyline level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative: If both C line and T line appear, the test indicates that the Amitriptyline level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

6.8.2 Thin Layer Chromatography - Amitriptyline AIM – Separation and identification of the Amitriptyline present in the sample.

6.8.2.1 Reagents and Materials Standard Amitriptyline, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

6.8.2.2 Method 6.8.2.2.1 Extraction procedure for Amitriptyline From urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

6.8.2.2.2 Extraction procedure for Amitriptyline From blood sample: Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

6.8.2.2.3 Sample application on TLC plate and development of chromatogram; Tank is saturated with the solvent for a minimum of 30 minutes. Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness) Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

6.8.2.3 Result Standard Amitriptyline gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Amitriptyline is present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

6.8.2.4 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

6.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

6.10Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 6.10.1  For Chromatographic Immuno assay Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

6.10.2 For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

6.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative.

6.12 Reportable interval of examination results. Same day 6.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

6.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (Positive or negative) along with the results of thin layer chromatography.

6.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe     disposal of waste materials including sample.

Diazepam 7.1 Purpose of the examination To screen for the presence of Diazepam

7.2 Principle of the procedure used for examinations; Colour tests followed by thin layer chromatography are performed on Gastric aspirate urine and blood samples.

7.2.1 Diazepam (Benzodiazepines) Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy –colored conjugate pad containing mouse antioxazepam antibodies coupled to colloidal gold, and 2) nitrocellulose membrane containing a Test (T) line and a Control (C) line. The Test line is coated with benzodiazepines –BSA, and the Control line is coated with goat anti-mouse IgG antibody. This test is a competitive binding immunoassay. The oxazepam in the urine specimen competes with the-BSA antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated antigen oxazepam antibodies. When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of oxazepam in the urine specimen is at or above the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of oxazepam in the urine specimen is at or above the cutoff, no Test line develops. The control line is coated with goat anti-mouse antibody, which should bind to the gold-antibody conjugate and form a burgundy color line regardless of the presence of oxazepam

7.2.2 Diazepam – Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer. For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

7.3 performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

7.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

7.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

7.6 Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

7.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

7.8 Procedural steps; 7.8.1 Diazepam (Benzodiazepines) –Chromatographic Immuno Assay

7.8.1.1 Precaution The instructions must be followed to obtain accurate results. •	Do not open the sealed pouch, unless ready to conduct the assay. •	Do not use expired devices. •	Dispose of all specimens and used assay materials as potentially biohazards.

7.8.1.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibriated to room temperature before testing.

•	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding the dropper vertically, add four drops of the specimen to the sample well marked as”S”on the device. •	Note: If migration is not observed in 30 seconds in the results window. Add one drop of urine specimen •	Read the test result between four (4) to seven (7) minutes after adding the specimen. •	Important: Do not read test results after seven (7) minutes. Interpretation of results:

C	      C	             C	  C              T	       T	             T	  T                  (+)                (-) Positive       Negative           Invalid Positive: If only the C line appears, the test indicates that the oxazepam level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative: If both C line and T line appear, the test indicates that the oxazepam level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

7.8.2 Diazepam Thin layer Chromatography AIM – Separation and identification of the Diazepam present in the sample. 7.8.2.1 Reagents and Materials Standard Diazepam, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

7.8.2.2 Method 7.8.2.2.1 Extraction procedure for Diazepam from urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish / petri dish. The ether layer in the dish is evaporated to dryness.

7.8.2.2.2 Extraction procedure for Diazepam From blood sample. Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

7.8.2.2.3 Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes  Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness). Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

7.8.2.2.4 Result Standard Diazepam gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Diazepam is present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

7.8.2.2.5 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

7.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

7.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 7.10.1 For Chromatographic Immuno assay Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

7.10.2 For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

7.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either possible on negative.

7.12  Reportable interval of examination results. Same day

7.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

7.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

7.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe      disposal of waste materials including sample.

7.16 Reference; 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation. 3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004. Morphine 8.1 Purpose of the examination To screen for the presence of Morphine

8.2  Principle of the procedure used for examinations; Chromatographic immuno assay followed by thin layer chromatography is performed on gastric aspirates urine and blood samples.

8.2 .1 Morphine  - Chromatographic immuno assay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy –colored conjugate pad containing mouse antimorphine antibodies coupled to colloidal gold, and 2) nitrocellulose membrane containing a Test (T) line and a  Control (C) line. The Test line is coated with morphine –BSA, and the Control line is coated with goat anti-mouse IgG antibody. This test is a competitive binding immunoassay. The oxazepam in the urine specimen competes with the morphine-BSA antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated anti morphine antibodies. When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of morphine in the urine specimen is at or above the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of morphine in the urine specimen is at or above the cutoff, no Test line develops. The control line is coated with goat anti-mouse antibody, which should bind to the gold-antibody conjugate and form a burgundy color line regardless of the presence of morphine.

8.2.2 Morphine-Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer. For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

8.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

8.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

8.5  Type of container and additives; No preservatives are used. Fresh clean containers are used.

8.6  Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

8.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

8.8	Procedural steps; 8.8.1 Chromatographic immuno assay 8.8.1.1 Precaution The instructions must be followed to obtain accurate results. •	Do not open the sealed pouch, unless ready to conduct the assay. •	Do not use expired devices. •	Dispose of all specimens and used assay materials as potentially biohazards. •	8.8.1.2 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibrated to room temperature before testing. •	Remove the test device with specimen identification. •	Holding the dropper vertically, add four drops of the specimen to the sample well marked as”S”on the device. •	Note: If migration is not observed in 30 seconds in the results window. Add one drop of urine specimen

Important: Do not read test results after seven (7) minutes.

8.8.1.3 Interpretation of results:

C 	      C	            C	  C              T	       T	            T	  T                  (+)                (-) Positive       Negative             Invalid Positive: If only the C line appears, the test indicates that the morphine level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative: If both C line and T line appear, the test indicates that the morphine level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

8.8.2  Thin Layer Chromatography AIM – Separation and identification of the Morphine present in the sample.

8.8.2.1 Reagents and Materials Standard Morphine, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

8.8.2.2 Method Extraction procedure for Morphine From urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish / petri dish. The ether layer in the dish is evaporated to dryness.

8.8.2.2.2 Extraction procedure for Morphine From blood sample Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

8.8.2.2.1 Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes. Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness). Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

8.8.2.3 Result Standard Morphine gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Morphine is present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

8.8.2.2	Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

8.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

8.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 8.10.1   For Chromatographic Immuno assay Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

8.10.2For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

8.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either possible on negative.

8.12 Reportable interval of examination results. Same day

8.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

8.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. A final interpretation is made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography. 8.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe     disposal of waste materials including sample. 8.16 Reference 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation. 3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004.

Paracetamol (Acetaminophen) 9.1 Purpose of the examination To screen for the presence of Paracetamol (Acetaminophen)

9.2 Principle of the procedure used for examinations; Colour tests followed by thin layer chromatography are performed on gastric aspirate, urine and blood samples.

9.2.1 Paracetamol – Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable absorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer. For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin

9.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

9.4 Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

9.5 Type of container and additives; No preservatives are used. Fresh clean containers are used.

9.6 Required equipment and reagents; Screening is done and interpretation is made manually. No    equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

9.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to     ensure reproducibility of results.

9. 8  Procedural steps; 9.8.1 Paracetamol- Qualitative Applicable to urine, stomach contents and scene residues.

9.8.1.1. Reagents: 1.	Concentrated HCI (relative density 1.18) 2.	Aqueous o-cresol solution (10 g/l) 3.	Aqueous ammonium hydroxide solution (4 mol/l)

9.8.1.2 Method 1.	Add 0.5 ml of HCl to 0.5 ml of sample, boil for 10 minutes and cool. 2.	Add 1 ml of o-cresol solution to 0.2 ml of the hydrolysate. 3.	Add 2 ml of ammonium hydroxide solution and mix for 5 seconds.

9.8.1.3 Result A strong, royal blue colour developing immediately indicates the presence of paracetamol. This test is very sensitive and will detect therapeutic dosage with paracetamol 24-48 hrs later.

9.8.1.4. Sensitivity P –aminophenol 1 mg/l

9.8.2  Thin Layer Chromatography AIM – Separation and identification of the Paracetamol present in the sample.

9.8.2.1 Reagents and Materials Standard Paracetamol, Solvent (Chloroform: Acetone, 4:1 & Methanol: Ammonia, 100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample.

9.8.2.2	Method 9.8.2.2.1	Extraction procedure for Paracetamol From urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish / petri dish. The ether layer in the dish is evaporated to dryness.

9.8.2.2.2	Extraction procedure for Paracetamol From blood       sample. Take 4 ml of serum sample add 2 ml of HCl (2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness. 9.8.2.2.3	Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes. Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness) Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization. 9.8.2.2.4 Result Standard Paracetamol gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Paracetamol is present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same) 9.8.2.2.5 Interpretation Made by comparing the Rf of the test sample (if any spots are   present) with that of standard solution. 9.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method. 9.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia)      and cross reactions; 9.10.1 For qualitative colour test Only aromatic amines, such as aniline, which also give rise to      p- aminophenol in urine after hydrolysis are known to interfere. Ethylenediamine (from aminophylline,) gives a green colour in this test. 9.10.2	For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required. 9.11 Principle procedure for calculating results, including      measurement uncertainty; No calculations are involved since result is given as either possible on negative. 9.12 Reportable interval of examination results. Same day 9.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test. 9.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. A final interpretation is made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography. 9.15 Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe disposal of waste materials including sample.

9.16  Reference 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with  the  United Nations Environment Programme and the International Labour Organisation. 3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004. Phenobarbitone 10.1 Purpose of the examination To screen for the presence of Phenobarbitone

10.2  Principle of the procedure used for examinations; Preliminary qualitative test using Barbiturates test cassette followed by thin layer chromatography is performed on gastric aspirate urine and blood samples.

10.2.1 Phenobarbitone-Chromatographic immunoassay This assay is a one-step lateral flow chromatographic immunoassay. The test strip includes 1) a burgundy-colored conjugate pad containing mouse anti-barbiturate antibodies coupled to colloidal gold; and 2) nitrocellulose membrane containing a Test (T) line and a Control (C) line. The test line is coated with barbiturate-BTG, and the Control line is coated with goat –anti -rabbit IgG antibody. This test is a competitive binding immunoassay. The barbiturate in the urine specimen competes with the barbiturate-BTG antigen coated on the nitrocellulose membrane for the limited binding sites of the conjugated anti-barbiturate antibodies. When an adequate amount of urine specimen is applied to the sample pad of the device, the urine specimen migrates by capillary action through the test strip. If the level of barbiturate in the urine specimen is below the cutoff (300 ng/ml), the Test line should appear as a visible burgundy line. If the level of barbiturate in the urine specimen is at or above the cutoff, no Test line develops. The C line binds to the gold-conjugated rabbit IgG forms a burgundy color line, regardless of the presence of barbiturate.

10.2.2	Phenobarbitone – Thin Layer Chromatography Thin layer chromatography is a multistage distribution process. This process involves solvents or solvent mixtures, sample molecules and a suitable adsorbent (mainly Silica). For TLC this adsorbent is applied to a suitable support like a glass plate, polyster or aluminium sheet in a thin layer. For the experiments with TLC, the mixture of substances to be separated is applied to a micro pre coated sheet and placed in to a development tank containing the development solvent. After completion of separation, placing the TLC plate in an Iodine chamber does visualization of chromatogram. The basic chromatographic measurement of a substance in TLC is Rf value, which is defined as

Rf  =   Distance the substance travels from the origin Distance the solvent front travels from the origin 10.3 Performance specifications (e.g. linearity, precision, accuracy expressed as uncertainty of measurement, detection limit, measuring interval, trueness of measurement; analytical sensitivity and analytical specificity); This is qualitative screening test that is detected as either positive or negative; hence parameters mentioned above in this section do not apply.

10.4  Primary sample collection (e.g. plasma, serum, urine); Blood (Plain 4-10 ml) Urine (50 ml) Gastric aspirate (10-25 ml)

10.5  Type of container and additives; No preservatives are used. Fresh clean containers are used.

10.6  Required equipment and reagents; Screening is done and interpretation is made manually. No equipments are needed for this purpose. Thin layer chromatography is done in a Chromatography chamber.

10.7 Calibration procedures (metrological traceability); Method is calibrated with known controls at regular intervals to ensure reproducibility of results.

10.8 Procedural steps; 10.8.1 Barbiturates  Chromatographic Immunoassay 10.8.1.2 Precaution •	The instructions must be followed exactly to obtain accurate results •	Do not open the sealed pouch, unless ready to conduct the assay •	Do not use expired devices •	Dispose of all specimens and used assay materials as potentially biohazardous.

10.8.1.3 Assay procedure •	Refrigerated specimens and other test materials, including devices, must be equilibrated to room temperature before testing. •	Remove the test device from pouch and place it on a flat surface. Label the device with specimen identification. •	Holding dropper vertically, add four drops of the specimens to the sample well •	Read the test result between four (4) to seven (7) minutes after adding the specimen. •	10.8.1.4 Interpretation of results. Important : Do not read test results after seven (7) minutes. The T line should be interpreted independently of the C line.

C	      C	            C	 C              T	       T	            T	  T                  (+)                (-) Positive        Negative               Invalid Positive: If only the C line appears, the test indicates that the barbiturates level in the sample is at a cutoff of 300ng/ml or higher Samples with positive results should be confirmed with a more specific method before a positive determination is made.

Negative If both C line and T line appear, the test indicates that the barbiturate level is below 300 ng/ml. Note: A faint T line should be considered negative.

Invalid: If no C line develops within 5 minutes, repeat the assay with a new test device.

10.8.2 Thin Layer Chromatography AIM – Separation and identification of the Barbiturate compound present in the sample.

10.8.2.1 Reagents and Materials Standard Barbiturate compound, Solvent (Chloroform:Acetone,4:1 & Methanol:Ammonia,100:1.5, prepared fresh), Chromatography tank, Silica gel coated TLC plate, Capillary tubes, TLC sprayer or Iodine chamber and sample. 10.8.2.2 Method 10.8.2.2.1 Extraction procedure for Phenobarbiturates from urine and gastric aspirate Take10 ml of sample in separating funnel; add few drops tartaric acid or phosphoric acid to it (pH: 3.5). Extracted with two 30 ml portions of diethyl ether. The extracts are combined and washed with 5ml of water. The washings are added to the sample. The diethyl ether layer is drained out and passed through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness. 10.8.2.2.2 Extraction procedure for Phenobarbiturates   from blood sample. Take 4 ml of serum sample add 2 ml of HCl(2mol/L) and 40 ml of Diethyl ether, shake vigorously for two minutes. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel. Diethyl ether layer is drain out and pass through  anhydrous sodium sulphate and collected in a china dish/ petri dish. The ether layer in the dish is evaporated to dryness.

10.8.2.2.3  Sample application on TLC plate and development of chromatogram Tank is saturated with the solvent for a minimum of 30 minutes. Reconstitute the extract in methanol. The stationary phase in TLC is aluminium sheet coated with silica gel (0.25 mm in thickness). Using capillary tubes, samples and standards are spotted twice, allowing the sample to dry in between spotting. Plate is placed on the tank for run to occur taking care that the plates are not tilted so that run will be in a straight line. Plate is removed from the tank when the solvent has moved till the desired solvent front. Plates are placed under fan for drying the plate. Once dried, placed in the Iodine chamber for visualization.

10.8.2.3 Result Standard Phenobarbitone gives spot on the plate with a characteristic Rf value, which will be constant for given conditions. If Phenobarbitone is present in the test sample, it will give a corresponding spot on the plate. (Rf value will be same)

10.8.2.4 Interpretation Made by comparing the Rf of the test sample (if any spots are present) with that of standard solution.

10.9 Quality control procedures; Samples, which are tested positive, are used as controls to test the method at regular intervals. Chromatography is done at very regular intervals to check the Rf with known standard solutions to validate the method.

10.10 Interferences (e.g., lipemia, haemolysis, bilirubinemia) and cross reactions; 10.10.1   For Chromatographic Immuno assay Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results. Varying range of urinary specific gravity and urinary pH does not affect the test results.

10.10.2	For Thin Layer Chromatography Chromatography of compounds that originate from biological extracts shows interferences from additional material present in sample extracts (matrix effects). For eg extracts of stomach contents may contain fatty materials and purification by extraction in to aqueous acid or alkali may be required.

10.11 Principle procedure for calculating results, including measurement uncertainty; No calculations are involved since result is given as either positive or negative.

10.12 Reportable interval of examination results. Same day

10.13 Alert/critical values, where appropriate, Any result, which is positive, is alerted immediately. Repeat analysis of a fresh urine sample is recommended before coming to a conclusion. Repeat analysis is considered part of the test.

10.14 Laboratory interpretation; Interpretation for each test is made as positive or negative. Final interpretations are made on the basis of individual test result (positive or negative) along with the results of thin layer chromatography.

10.15	Safety precautions; Sample and reagents are handled with utmost care. Care is taken for safe  disposal of waste materials including sample.

10.16	References 1.Directorate of Forensic Science, Laboratory procedure Manual for  Toxicology. 2. R.J Flanagan and et al. Basic Analytical Toxicology published By WHO in Collaboration   with the United Nations Environment Programme and the International Labour Organisation. 3.Clarke’s Analysis of Drugs and Poisons, 3rd Edn.The Pharmaceutical Press, London, 2004.