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Evaluation of an enzymatic method for the measurement of uric acid
98 / P10
1982 CSCC ABSTRACTS
The stock reagent contains 0.5 mol/L glycine, 0.4 mol/L hydrazine and brought to pH 9.1 with NaOH (-20g pellets/L buffer). For working reagent mix 150 mg NAD and 20 uL LD (Boehringer Mannheim Cat. NO. 107077) with 25 mL stock reagent. The sample fraction is 1% of the reagent volume e.g. 10 uL plasma mixed with 1 ml working reagent. In%mediately after sample and reagent are combined t they are introduced into the curet, preincubated for 10 sec and ~ A is measured over the next 30 sec. The blank reaction consists of a sample of water with reagent r the calibrate is a solution Of 5 mmol/L lactate. This method is linear to about i0 ~ o l / L lactate. Sample dilution may be required for higher concentrations of lactate. For a lactate Q.C. sample at 1.8 mabel/L, within run CV was 5.8%, between run CV was 6.2%. Mean recovery Of 2 su~ol/L extra lactate added to samples with initial lactates of 0.4 to iI ~tmol/L was 90%. One can adapt this method to different timing constraints by using the formula, Vopt = Km/tm as described by Atwood and DiCesare ((1975) Clin. Chem. 21, 1263). For the LD preparation as used here the effective Vmax in the working reagent was 0.5 U per uL LD added and the Km was 0.69"umol/mL.
50 SIMULTANEOUS QUANTITATION OF ~ T H O T N E X A T E AND 7-HYDROXY~O~REXATE BY HPLC. Collier r C.P. and ~ i d i n , S.J., Dept. Clin. Biochem. and Pharmacol, Univ. Of Toronto, and Res. Inst., Hosp. for Sick Children, Toronto, Ontario MbG Ixg. Following high-dose methotrexate (MTX) therapy, leucovorin rescue should be continued until the concentration of b~X is less than 10-7'10 -8 M. Below this concentration the risk O f drugrelated toxic)ties, such as myelosuppression, is minimal. We have developed an higb performance liquid chromatographic (IIPLC) assay with UV spectrophotometric detection (305 nm) for the simultaneous quantitation of MTX and its putatively nephrotoxic 7-hydroxy me tabolite (7OH-MTX) in serum. The serum sample (300 ul) and internal standard, S-chlorotheophylline (geT), are applied to SEP-PAK R C-18 cartridges (Waters ASSOC.) in 5 ml Of 0.2 M acetate buffer, pll 5.0. After a wash of I0 ml 1120, the compounds of interest are eluted from the cartridges in 2 ml methanol. The methanol is evaporated and the residue is reconstituted in 5 mM HCI before injection. The mobile phase is a 85/15 ratio of I0 mM phosphate buffer, pH 4.5/acetonitrile. Under these conditions, 7OH-MTX elutes first followed by 8CT and then MTX. Recovez> and precision data are sur~narized: % Recovery (n=lb) Between-day Precision(n=20) Concentration(uM) % Recovery ConeentraTion(uM) %CV MTX 2.31 78.3 2.76 8.6 0.23 84.9 0.25 15.1 7OH-MTX 4.35 67.6 4.40 8.9 0.48 72.8 0.53 7.6 A comparison of our HPLC procedure with EMIT R (Syva Co.) and a radiochemical ligand binding (CPB) assay for MTX (Anal. Biochem. 70, 54-63, 1976) produced the following results: Y X n Slope Intercept r HPLC CPB 7--2 0.85 3.19 0.~65 HPLC EMTT R (<100 RM) 29 0.96 0.17 0.984 HPLC EMIT E (>100 sM) 25 0.60 45.42 0.882
52 EVALUATION OF AN ENZYmaTIC METHOD ~OR ~ [ E MEASUREMENT OF URIC ACID. Theresa Verzuu and Marie D'Costa, Dept. of Laboratories, Mount Sinai Rospltal, Toronto, Ontario, MbG IX$. We evaluated the AGENT TM reagent for the measurement of uric acid in se~um and urine. The procedure is based on the reactions: Uric acid + 02 + H20 11202 + ethanol
We d e v e l o p e d a sensitive and specific assay for measurement of morphine in serum by HPLC with electrochemical detection (Ther. Drug Monit., in press). We now report a procedure for analysis of morphine-3-glucuronide (M-3-GL), the major metabolite of morphine in serum. To i00 sl serum add 50 3~I of a 1 M potassium acetate buffer pH 5.00, containing the internal standard, 5-hydroxyquinoline. Subsequently add 20 U1 8-glucuronidase dissolved ~n distilled water (i mg enzyme per 20 HI is equivalent to 919 U/mg). After mixing, incubate the capped tubes at 37°C for 4 h or at room temperature for 24 h. Following incubation alkalinize the serum by adding 21.8 ~l i N ~ O H . ExtKaction a n d c h r o m a t o g r a p h y of total morphine (morphine and morphine released from M-3-GL) follows the procedure for morphine described previously (above reference). The conversion of M-3-GL to morphine is virtually complete. The recovery Of morphine is shown in the table below. Concentration(up~L) % Recovery Precision %CV(n=ll) Morphine 250 74 6.2 i00 71 8.0 25 73 6.0 X Y % Recovery Precision %CV(n=ll) Morphine (X) . 250 + 261 72 8.4 + M-3-GL (Y) i00 + 104.4 74 8.9 25 + 52.2 75 9.1 Amount of 5-hydroxyquinoline added 6.5 ng 64 8.8(n=ii) Tne metabolite concentration is calculated as fol~ows: concentration of M=3-GL = (total morphine cone. - morphine cone.) x 1.6173 (i.8173 = M.Wt. M-3-GL/M.Wt. M). The pharmaeokinetics of morphine ~Lnd its major metabolite M-3-GL are being studied in children.
allantoin + CO 2 + ]{202
acetaldehyde + H20 alcohol ) dehydrogenase
acetaldehyde + NADH + H +
ethanol + NAD +
The analysis was performed on the ABA-100 in the ERR mode at 30OC with a 340/380 filter and a 1:26 syringe plate. At 37°C the results were the same. NBS uric acid standards (238-595 pmol/l) in Li2CO3 were used for calibration. A candidate reference method (P. Duncan et al., 1980, USHEW PHS, CDC, Georgia) based on analysis of deprotelnised samples at 283 nm was used as the reference method. The reconstituted enzyme reagent was stable for 3 days at 4Oc. Urine specimens were diluted 1:5. Between-day precision for the AGENT TM reagent was 2.2% CV, 2.6% CV and 1.9% CV at 256, 405 and 571 Hmol/l serum uric acid respectively (n=22). Linearity and detection limits were 950 and 15 ~mol/l respectively. Recovery of uric acid added to serum pools and urine was 98-103%. Comparison of 118 patients' sere with the reference method (x) gave a regression analysis of y=l.021x + 5.2, Sy/X=15.5, r=0.9972. Urine samples also correlated well. Lipemia (trlg!yceride up to 9 mmol/l} did not significantly interfere. Moderate hemolysis (hemoglobin up to 5 g/l) had no effect; however, severe hemolysis caused spurious elevations in the results. The addition of bilirubln (214 Hmol/l) had no effect but higher levels increased the value of measured uric acid. We conclude that the AGENT TM uric a c i d r e a g e n t has adequate precision and accuracy for routine measurements of uric acid on the ABA-100.
53 EVALUATION OF A SUBSTKATE-I.ABELED FLUORESCENT IHMUNOASSAY (SLFIA) FOR THE [)ETERHINATION OF QUlNIDINE IN SERUM Sur[a, D_u. and Wri8h~, L.A., D e p t . Clin. Biochem. The %dellesley }lospi'tal, Toronto, Ontario M4Y IJ3 In view of the potential harmful effect of benzene used in ti~e double extraction f]uorescence (DEF) procedure for the determination oF quinidine in serum, the SLF[A (Ames TDA TM Quinidine) was evaluated in terms of accuracy, precision and reagent loT-to-lot variability. Recovery studies indicate that both procedures were not interfered with by most commonly prescribed drugs except for Triamterene which erroneously elevate quinidine levels and for dipycidamole w h i c h also elevate ['lie SLF[A procedure only. A summary of the day-t0-day precision of controls and iot-toio~ variability of two groups of 50 clinical specimens, analyzeu twice, is g~ven as follows: Ames Controls A B D
51 DETERMINATION OF MORPHINE AND MORPHINE-3-GLUCUBONIDE IN SERUM. Vandenberghe, H.M. and Soldin, S.J., Dept. Clin. Biochem. and Phazlnacol., Univ. of Toronto, and Res. Inst., NosiL for Sick Children, Toronto, Ontario MbG IX8.
uricase)
Catalase)
DEF Hean (umol/L) 20 6.3 20 12.5 20 24.6 N
C.V. 3.30 3.37 2.79
8LFIA (Lot Mean (umol/L) l0 6.a lO 11.9 l0 24.4
N
I) C.V. 8.1] 3.70 3.23
SLFiA (Lot t D ~ Mean C.V. (umollL) I0 6.5 8.69 I0 12.4 6 . 5 1 l0 24.3 2.88
Clinical Specimens Crou~ I : SLFIA= -).04 - 1.098 DEF. .I=49; r=0.98, t=l.0]8 Crou~ II : SLFIA= -].30 - 1.223 DEF. n=50; r=0.98, t=5.6]9 In conclusion The SLFIA procedure is simple ro perform and produces mea~ quality control values comparable re The DEE procedure althou8h ar The lower levels of quinidlne used t h e SLFIA procedure tended to produce greater coefficient of variation. A degree Of lot-to-lot varlability of the SLFIA procedure was observed.
~4 COMPARISON OF COLUMN CHROMATOGRAPHIC, ELECTROPHORETIC AND CHEMICAL DETERMINATIONS OF GLYCOSYLATED IIEMOGLOBIN (HbAi). R.2. Yatscoff. St. Joseph's Hospital, London, Ontario & DIV of Clinical B~ochemistry, University of Western Ontarlo. q~wo ~ini-column methods (BioRad & Helena) were compared wlth an electrophoretic [Corning) and chemical method (thiobarbituric acid) for the determination of glycosylated hemoglobin. The precislon studies of these methods were as follows. BIORAD HELENA EPHOR CHEMICAL INTRA RUN
Level L H
N CV% 5 6.5 I0 10.9
N CV% 5 1.31 i0 3.35
N 8 8
CV% 5.2 6.6
N I0 i0
CV% 3.0 2.2
Level N CV% ~ CV% N CV% N CV~ INTER L 13 16.9 14 7.8 20 8.9 IO 13.7 RUN H 8 16.5 II 10.4 37 8.5 I0 10.4 Correlation studies showed that the electrophoretic method correlated the best with the BioRad columns (r=0.91 n=70] followed oy the Helena column (r=0.91 n=63] and the chemical method (r=0.865 n=20). The effects of preincubation to remove the labile glycosyrated hemoglobin in both the electrophoretic and BioRad column method was investigated. Preincubation had no statistically slgnificanr effect [p >0.05) or the glycosylated hemoglobin
1982 CSCC ABSTRACTS
The stock reagent contains 0.5 mol/L glycine, 0.4 mol/L hydrazine and brought to pH 9.1 with NaOH (-20g pellets/L buffer). For working reagent mix 150 mg NAD and 20 uL LD (Boehringer Mannheim Cat. NO. 107077) with 25 mL stock reagent. The sample fraction is 1% of the reagent volume e.g. 10 uL plasma mixed with 1 ml working reagent. In%mediately after sample and reagent are combined t they are introduced into the curet, preincubated for 10 sec and ~ A is measured over the next 30 sec. The blank reaction consists of a sample of water with reagent r the calibrate is a solution Of 5 mmol/L lactate. This method is linear to about i0 ~ o l / L lactate. Sample dilution may be required for higher concentrations of lactate. For a lactate Q.C. sample at 1.8 mabel/L, within run CV was 5.8%, between run CV was 6.2%. Mean recovery Of 2 su~ol/L extra lactate added to samples with initial lactates of 0.4 to iI ~tmol/L was 90%. One can adapt this method to different timing constraints by using the formula, Vopt = Km/tm as described by Atwood and DiCesare ((1975) Clin. Chem. 21, 1263). For the LD preparation as used here the effective Vmax in the working reagent was 0.5 U per uL LD added and the Km was 0.69"umol/mL.
50 SIMULTANEOUS QUANTITATION OF ~ T H O T N E X A T E AND 7-HYDROXY~O~REXATE BY HPLC. Collier r C.P. and ~ i d i n , S.J., Dept. Clin. Biochem. and Pharmacol, Univ. Of Toronto, and Res. Inst., Hosp. for Sick Children, Toronto, Ontario MbG Ixg. Following high-dose methotrexate (MTX) therapy, leucovorin rescue should be continued until the concentration of b~X is less than 10-7'10 -8 M. Below this concentration the risk O f drugrelated toxic)ties, such as myelosuppression, is minimal. We have developed an higb performance liquid chromatographic (IIPLC) assay with UV spectrophotometric detection (305 nm) for the simultaneous quantitation of MTX and its putatively nephrotoxic 7-hydroxy me tabolite (7OH-MTX) in serum. The serum sample (300 ul) and internal standard, S-chlorotheophylline (geT), are applied to SEP-PAK R C-18 cartridges (Waters ASSOC.) in 5 ml Of 0.2 M acetate buffer, pll 5.0. After a wash of I0 ml 1120, the compounds of interest are eluted from the cartridges in 2 ml methanol. The methanol is evaporated and the residue is reconstituted in 5 mM HCI before injection. The mobile phase is a 85/15 ratio of I0 mM phosphate buffer, pH 4.5/acetonitrile. Under these conditions, 7OH-MTX elutes first followed by 8CT and then MTX. Recovez> and precision data are sur~narized: % Recovery (n=lb) Between-day Precision(n=20) Concentration(uM) % Recovery ConeentraTion(uM) %CV MTX 2.31 78.3 2.76 8.6 0.23 84.9 0.25 15.1 7OH-MTX 4.35 67.6 4.40 8.9 0.48 72.8 0.53 7.6 A comparison of our HPLC procedure with EMIT R (Syva Co.) and a radiochemical ligand binding (CPB) assay for MTX (Anal. Biochem. 70, 54-63, 1976) produced the following results: Y X n Slope Intercept r HPLC CPB 7--2 0.85 3.19 0.~65 HPLC EMTT R (<100 RM) 29 0.96 0.17 0.984 HPLC EMIT E (>100 sM) 25 0.60 45.42 0.882
52 EVALUATION OF AN ENZYmaTIC METHOD ~OR ~ [ E MEASUREMENT OF URIC ACID. Theresa Verzuu and Marie D'Costa, Dept. of Laboratories, Mount Sinai Rospltal, Toronto, Ontario, MbG IX$. We evaluated the AGENT TM reagent for the measurement of uric acid in se~um and urine. The procedure is based on the reactions: Uric acid + 02 + H20 11202 + ethanol
We d e v e l o p e d a sensitive and specific assay for measurement of morphine in serum by HPLC with electrochemical detection (Ther. Drug Monit., in press). We now report a procedure for analysis of morphine-3-glucuronide (M-3-GL), the major metabolite of morphine in serum. To i00 sl serum add 50 3~I of a 1 M potassium acetate buffer pH 5.00, containing the internal standard, 5-hydroxyquinoline. Subsequently add 20 U1 8-glucuronidase dissolved ~n distilled water (i mg enzyme per 20 HI is equivalent to 919 U/mg). After mixing, incubate the capped tubes at 37°C for 4 h or at room temperature for 24 h. Following incubation alkalinize the serum by adding 21.8 ~l i N ~ O H . ExtKaction a n d c h r o m a t o g r a p h y of total morphine (morphine and morphine released from M-3-GL) follows the procedure for morphine described previously (above reference). The conversion of M-3-GL to morphine is virtually complete. The recovery Of morphine is shown in the table below. Concentration(up~L) % Recovery Precision %CV(n=ll) Morphine 250 74 6.2 i00 71 8.0 25 73 6.0 X Y % Recovery Precision %CV(n=ll) Morphine (X) . 250 + 261 72 8.4 + M-3-GL (Y) i00 + 104.4 74 8.9 25 + 52.2 75 9.1 Amount of 5-hydroxyquinoline added 6.5 ng 64 8.8(n=ii) Tne metabolite concentration is calculated as fol~ows: concentration of M=3-GL = (total morphine cone. - morphine cone.) x 1.6173 (i.8173 = M.Wt. M-3-GL/M.Wt. M). The pharmaeokinetics of morphine ~Lnd its major metabolite M-3-GL are being studied in children.
allantoin + CO 2 + ]{202
acetaldehyde + H20 alcohol ) dehydrogenase
acetaldehyde + NADH + H +
ethanol + NAD +
The analysis was performed on the ABA-100 in the ERR mode at 30OC with a 340/380 filter and a 1:26 syringe plate. At 37°C the results were the same. NBS uric acid standards (238-595 pmol/l) in Li2CO3 were used for calibration. A candidate reference method (P. Duncan et al., 1980, USHEW PHS, CDC, Georgia) based on analysis of deprotelnised samples at 283 nm was used as the reference method. The reconstituted enzyme reagent was stable for 3 days at 4Oc. Urine specimens were diluted 1:5. Between-day precision for the AGENT TM reagent was 2.2% CV, 2.6% CV and 1.9% CV at 256, 405 and 571 Hmol/l serum uric acid respectively (n=22). Linearity and detection limits were 950 and 15 ~mol/l respectively. Recovery of uric acid added to serum pools and urine was 98-103%. Comparison of 118 patients' sere with the reference method (x) gave a regression analysis of y=l.021x + 5.2, Sy/X=15.5, r=0.9972. Urine samples also correlated well. Lipemia (trlg!yceride up to 9 mmol/l} did not significantly interfere. Moderate hemolysis (hemoglobin up to 5 g/l) had no effect; however, severe hemolysis caused spurious elevations in the results. The addition of bilirubln (214 Hmol/l) had no effect but higher levels increased the value of measured uric acid. We conclude that the AGENT TM uric a c i d r e a g e n t has adequate precision and accuracy for routine measurements of uric acid on the ABA-100.
53 EVALUATION OF A SUBSTKATE-I.ABELED FLUORESCENT IHMUNOASSAY (SLFIA) FOR THE [)ETERHINATION OF QUlNIDINE IN SERUM Sur[a, D_u. and Wri8h~, L.A., D e p t . Clin. Biochem. The %dellesley }lospi'tal, Toronto, Ontario M4Y IJ3 In view of the potential harmful effect of benzene used in ti~e double extraction f]uorescence (DEF) procedure for the determination oF quinidine in serum, the SLF[A (Ames TDA TM Quinidine) was evaluated in terms of accuracy, precision and reagent loT-to-lot variability. Recovery studies indicate that both procedures were not interfered with by most commonly prescribed drugs except for Triamterene which erroneously elevate quinidine levels and for dipycidamole w h i c h also elevate ['lie SLF[A procedure only. A summary of the day-t0-day precision of controls and iot-toio~ variability of two groups of 50 clinical specimens, analyzeu twice, is g~ven as follows: Ames Controls A B D
51 DETERMINATION OF MORPHINE AND MORPHINE-3-GLUCUBONIDE IN SERUM. Vandenberghe, H.M. and Soldin, S.J., Dept. Clin. Biochem. and Phazlnacol., Univ. of Toronto, and Res. Inst., NosiL for Sick Children, Toronto, Ontario MbG IX8.
uricase)
Catalase)
DEF Hean (umol/L) 20 6.3 20 12.5 20 24.6 N
C.V. 3.30 3.37 2.79
8LFIA (Lot Mean (umol/L) l0 6.a lO 11.9 l0 24.4
N
I) C.V. 8.1] 3.70 3.23
SLFiA (Lot t D ~ Mean C.V. (umollL) I0 6.5 8.69 I0 12.4 6 . 5 1 l0 24.3 2.88
Clinical Specimens Crou~ I : SLFIA= -).04 - 1.098 DEF. .I=49; r=0.98, t=l.0]8 Crou~ II : SLFIA= -].30 - 1.223 DEF. n=50; r=0.98, t=5.6]9 In conclusion The SLFIA procedure is simple ro perform and produces mea~ quality control values comparable re The DEE procedure althou8h ar The lower levels of quinidlne used t h e SLFIA procedure tended to produce greater coefficient of variation. A degree Of lot-to-lot varlability of the SLFIA procedure was observed.
~4 COMPARISON OF COLUMN CHROMATOGRAPHIC, ELECTROPHORETIC AND CHEMICAL DETERMINATIONS OF GLYCOSYLATED IIEMOGLOBIN (HbAi). R.2. Yatscoff. St. Joseph's Hospital, London, Ontario & DIV of Clinical B~ochemistry, University of Western Ontarlo. q~wo ~ini-column methods (BioRad & Helena) were compared wlth an electrophoretic [Corning) and chemical method (thiobarbituric acid) for the determination of glycosylated hemoglobin. The precislon studies of these methods were as follows. BIORAD HELENA EPHOR CHEMICAL INTRA RUN
Level L H
N CV% 5 6.5 I0 10.9
N CV% 5 1.31 i0 3.35
N 8 8
CV% 5.2 6.6
N I0 i0
CV% 3.0 2.2
Level N CV% ~ CV% N CV% N CV~ INTER L 13 16.9 14 7.8 20 8.9 IO 13.7 RUN H 8 16.5 II 10.4 37 8.5 I0 10.4 Correlation studies showed that the electrophoretic method correlated the best with the BioRad columns (r=0.91 n=70] followed oy the Helena column (r=0.91 n=63] and the chemical method (r=0.865 n=20). The effects of preincubation to remove the labile glycosyrated hemoglobin in both the electrophoretic and BioRad column method was investigated. Preincubation had no statistically slgnificanr effect [p >0.05) or the glycosylated hemoglobin