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Identification and analysis of tris(-butoxyethyl) phosphate by a dual-column and dual-detector GLC procedure
98 / P5
1982 CSCC ABSTRACTS ( C o r n l n g M e d i c a l , M e d f l e l d , MA 0 2 0 5 2 ) . The isoenzyme z o n e s w e r e q u e s t | t a r e d by d e n s i t o m e t r i c s c a n n i n g on Beckman Microzone Densltemeter. Erythrocyte lysates w e r e p r e p a r e d as d e s c r i b e d in r e f e r e n c e s ] and 2. In healthy indlviduals~ the r a t i o in s e r u m w a s less than |.0 ( M ± I S D = 0 . 7 1 ± 0 . 2 0 ) . In e r y t h r o c y t e l y s a t e / o f the same individuals, the r a t i o v a r i e d from less than l.O to g r e a t e r than 1.0 ( M ± I S D = ] . 0 9 ± 0 . 1 5 ) . In p a t i e n t s w i t h a c u t e m y o c a r d i a l i n f a r c t i o n or e v i d e n c e of | s i t e v a s c u l a r h e m o l y s i s , the r a t i o in s e r u m w a s g r e a t e r than I.D. In v i t r o h e m o l y s i s , i n d u c e d by a d d i n g h e m o l y s a t e to s e r u m , i n c r e a s e d the r a t i o in the serum. However w h e t h e r h e m o l y s i s p e r se c a u s e d the ratio to " f l i p " dep e n d e d on d e g r e e of h e m o l y s i s and i n i t i a l v a l u e of the r a t i o in the s e r u m and e t y t h r o c y t e lysate. The results s h o w that h e m o l y s i s can c a u s e a r e v e r s a l of L D - I / L D - 2 r a t i o in s e r u m by this m e t h o d . ThereFore a "flipped" r a t i o in c a r d i a c i s o e n z v m e r e s u l t s must be i n t e r p r e t e d with caution.
20 AMNIOTIC H~UID PHOSPHOLIPIDS QUANTITATED ON HICI{ EFFICIENCY THIN LAYER CHROMATOCRAPHY PLATES. Hayes~ A.N. and Hindmarsh, J.T., Biochemistry Division, Dept. oF Lab Medicine, Ottawa Civic Hospital, Ottawa, Ontario K~Y 4E9. Current interest In measuring not only amniotlc fluid lecithin/ sphingomyelin ratios but dlsaturated phosphatidyl choline and phosphatidyl glycerol quantitatfon brings problems which cannot always be solved by consulting current literature. It may be difficult to duplicate published results because of the need to use a different supplier of TLC plates and densltometer scanner, as well as different visualization techniques. A method is described for using High Efficiency TiC plates (Analteoh HEq%C-HL) with an adjusted Chtoroform/(Methanol)/N}[~0H developing solvent which gave good separation of all common amniotic fluid phospholipld components except phosphatidyl serine in 7.5 cms a n d a fifteen minute running time. Visualization was by fluorescence produced after spraying with 8-anllino-1naphthalene sulfonic acid (ANSA) (Heyneman, R.A. et al (1972) d. Chromatogr. 68, 285). The increased sensitivity of this reagent versus common charring methods iS an advantage for measurement of the low phosphatidy] glycerol concentrations of interest. Pre-treatment of plates with cupric solution removes phosphatldyl serlne interference as It renders It non-fluorescent with the ANSA spray. Estimation can he made by visual comparison with standards or by scanning plates on Beckman Model CDS-200 Computing Densitometer System in fluorescence mode. Results are presented for linearlty of fluorescent response for phosphatidyl glycerol, lecithin, sphingomyeIin, and disaturated phosphatidyl choline in the range of clinical interest and for some typical amniotic fluids.
dard [2-(4-benzoylphenyl)-butyric acid, L.~.], a double extraction procedure is performed. The first step involves acidification with 0.5 ,~L of HCI I mol/L and extraction in an organic phase (9.5 mL of an hexane/iso-amyl alcohol mixture, 99/I). l, the second step, drug and I.S. are extracted from the organic phase with 1.0 ~uL of a phosphate buffer, 30 ~ o l / L , pH 8.0. We inject 250 uL of the aquous phase. The reverse phase chromatographic conditions are: stationary phase, C18 bonded phase; mobile phase, 50% acetonltrile, 50% phosphate buffer 45 mmol/L, pH 3.0; flow rate, 2.0 mL/mln. The detector is set at a wavelength of 254 nm and at 0.05 absorbance units full scale. Average retention times are 5.9 min for l.S. and 8.8 min for indomethacin. In serum, no peak was found to interfere with indomethacln. Linearity was observed up to 5 mg/L. The lower limit of detection was 0.3 mg/L but, if needed, it could be easily lowered to 0.05 mg/L. The results of precision and accuracy studies are presented in the following table: Drug added (m$/L)
Within assay mean (m~/L)
0.7 1.5 3.0
0.71 1.53 3.06
PRECISION C.V. Between assay (%) mean (mB/L~
C.V. (%)
ACCURACY % Recovery ± i S.D.
4.3 1.6 1.8
7.3 6.8 6.1
98.9t 7.2 I01.2± 6.9 101.0± 6.21
0.69 1.52 3.03
Preliminary data indicate tha~ other anti-inflammatory drugs (including ¢arprophen, ibuprophen, naproxen and ketoptophen) could be measured with this same method (or minor modifications).
2~ IDENTIFICATION AND ANALYSIS OF TRIS(-BUTOXYET}~L) PHOSPHATE BY A DUAL-COLUMN AND DUAL-DETECTOR CLC PROCEDURE Suria~ D. and Wright, L.A. Dept. Clin. Biochem. The Wessesley Hospital, Toronto, Ontario M4Y 133 Trls(-butoxyethyl)phosphate (TBEP), a constituent of stoppers in certain blood collecting tubes, has been shown to lower the plasma level of some basic drugs spuriously (gorge. O. et.al. (|977) Clin. Pharmacol, Therap. 22:539). Until these stoppers can be completely eliminated an attempt has been made to identify TBEP in the blood sample or stopper and to assess its possible laterference with the analysis of drugs by means of gas chromatography (GO). A Hewlett-Packard 5880 GC equipped with a 3.8% SE-30 column/ FID detector and a 3% SP-2250 column/NP detector was used. The analyses were performed simultaneously on both columns at [sothermlc oven temperature set at 250 C. TBEP (Aldrich 13,059-]) was detected as a single peak on the SE-30 system at a retention time (RT) Of approximately 2.5 min, and as three peaks on the SP-2250 column system at RT 0.6, 1.4 and 4.5 min. TBEP has been found to cause a shift of disopytamide, quinidine, lidoealne, procainamide and propranolol into the erythrocyte, thus lowering the plasma level. In add|flea, it will interfere with drugs having the retention times in the region of those given above.
21
THE USE OF SILICA SEP-PAE C A / ~ I D ~ FOR PURIFICATION OF CHOLESTERYL ESTERS FR~I HUMAN SERiaL WANG, S.T., and Peter, F., Biochemistry Laboratory, Laboratory Services Branch, Ontario Ministry of l)ealth, Toronto, Ontario M~q 5K9. A simple and fast analytical procedure for separation and purification of cholesteryl esters of human serum is described: A single lipid extract, together with cholesteryl pentadecanoate, as an internal standard, was passed through a silica SEP-PAK (Waters Scientific) and was eluted with 1.5% ethyl ether in petroleum ether. With this solvent system, only cholesteryl esters were eluted from the coltm~. The separation was verified with TIC system On silica gel with petroleum ether/e~]yl ether/glacial acetic acid (80/20/]) as a solvent. It showed a very clean TLC chromatogram of cholesteryl esters without any additional spots. The cbolesteryl esters were quantified by analyzing their fatty acid c c ~ s i t i o n as methyl esters by GLC. The reprOducibility of the methOd was estimated by carrying out six replicate analyses on one serum sample. The coefficient of variatic~ was 0.8-2.9% for the major fatty acids (C16:0, C18:1 and C18:2) and 2.0-]6.6% for the minor fatty acids examined, (C16:1, C18:0, C20:0 and C20:4). The recovery was determined by adding a known amount of cholesteryl ester standard of cholesteryl palm|tare, cholesteryl oleate and cholesteryl linofeats to a serum s ~ o l e , then assaying the sample with the method. The percent recoveries for cholesteryl palm|rate, cholesteryl oleate and cholesteryl linoleate were 90.7, 92.3 and 91.0% respectively. We have evaluated the usefulness of the SEP-PAK cartridges method over ~ e conventional TIC method for the clean up of the lipid extract of serum samples for the analysis of cholesteryl esters. The method is suitable for the measurement of both cholesteryl esters of the total and HDL fraction in small serum sanples.
22
SERUM INDOMETHACIN DETER}IINATION BY HIGH PRESSURE LIQUID CHROMATOGRAPHY. Leclercr P., Billon, B., and PetitClerc, C., Centre Hospitaliet Universitalre de Sberbrooke, Sherbrooke, Quebec JIH 5N4 We present and evaluate a HPLC method for the measurement of Isdomethacin in serum. After adding 2 mg/L of the internal stan-
2q ADAPTATION OF THE BMC ENZYMATIC CREATININE TO THE ILMULTISTAT III: CLINICAL EVALUATION. Sold|n, S.J. and McPherson, K.A., Dept. Clin. Biochem. and Pharmacol., Univ. of q~Dronto, and Res. Inst., Heap. for Sick Children, Toronto, Ontario MbG IX8. The enzymatic treat|nine procedure employing treat|nine amidohydrolase has been well documented in the literature (Wahlefeld, A.W. et el. (1972) Scand. J. Clin. Lab. Invest. 29, Suppl. 126, Abstract ,~30.i and Jaynes, P. et al. (1981) Clin. Chem. 27, 1034, Abstract ~052). We have adapted the Boehringer Mannheim p r O C e dure to the IL-Multistat llI. blultistat settings used were: T=30°C, ~=340 am, delay = 3s, interval = 540s. Loader settings were: sample volume 20 ul (40 ul flush), reagent volume I00 lJl (10 lJl flush), blank switch on diluent. The reagent mix used was 4.95 ml reagent i, 0.15 ml reagent 2, 0.30 ml reagent 3, and either 75 ul of reagent 4 or 75 ul of water. (The latter is used for the blank run). Results for standards, controls and samples are calculated by plotting &A (=AAcreatininase - ~Awater - AAreagent blank) vs. creatinine concentration. The between-day precision at serum concentrations of 4.2, 8.4 and 28.9 mg/L qave coefficients of variation of 14.1%, 7.2% and 2.5% respectively (n=2O). Addition of 20, 40 and 66.6 mg/L oF creatinlne to a plasma pool resulted in recoveries of 100%, 99% and 95% respectively (n=lO) . Patient Population n ~ ~ r Slope Intercept BbIC Beckman Premature and fullterm infants 72 0.65 0.80 O.9199 1.162 O.04R Nephrology excluding dialysis patients 69 2.28 2.37 0.9859 1.056 0.040 Dialysis patients 20 7.66 8.54 0.9536 0.8964 1.673 The 95th percentile for the premature and full-term infants was found to be 10.6 mg/L.
1982 CSCC ABSTRACTS ( C o r n l n g M e d i c a l , M e d f l e l d , MA 0 2 0 5 2 ) . The isoenzyme z o n e s w e r e q u e s t | t a r e d by d e n s i t o m e t r i c s c a n n i n g on Beckman Microzone Densltemeter. Erythrocyte lysates w e r e p r e p a r e d as d e s c r i b e d in r e f e r e n c e s ] and 2. In healthy indlviduals~ the r a t i o in s e r u m w a s less than |.0 ( M ± I S D = 0 . 7 1 ± 0 . 2 0 ) . In e r y t h r o c y t e l y s a t e / o f the same individuals, the r a t i o v a r i e d from less than l.O to g r e a t e r than 1.0 ( M ± I S D = ] . 0 9 ± 0 . 1 5 ) . In p a t i e n t s w i t h a c u t e m y o c a r d i a l i n f a r c t i o n or e v i d e n c e of | s i t e v a s c u l a r h e m o l y s i s , the r a t i o in s e r u m w a s g r e a t e r than I.D. In v i t r o h e m o l y s i s , i n d u c e d by a d d i n g h e m o l y s a t e to s e r u m , i n c r e a s e d the r a t i o in the serum. However w h e t h e r h e m o l y s i s p e r se c a u s e d the ratio to " f l i p " dep e n d e d on d e g r e e of h e m o l y s i s and i n i t i a l v a l u e of the r a t i o in the s e r u m and e t y t h r o c y t e lysate. The results s h o w that h e m o l y s i s can c a u s e a r e v e r s a l of L D - I / L D - 2 r a t i o in s e r u m by this m e t h o d . ThereFore a "flipped" r a t i o in c a r d i a c i s o e n z v m e r e s u l t s must be i n t e r p r e t e d with caution.
20 AMNIOTIC H~UID PHOSPHOLIPIDS QUANTITATED ON HICI{ EFFICIENCY THIN LAYER CHROMATOCRAPHY PLATES. Hayes~ A.N. and Hindmarsh, J.T., Biochemistry Division, Dept. oF Lab Medicine, Ottawa Civic Hospital, Ottawa, Ontario K~Y 4E9. Current interest In measuring not only amniotlc fluid lecithin/ sphingomyelin ratios but dlsaturated phosphatidyl choline and phosphatidyl glycerol quantitatfon brings problems which cannot always be solved by consulting current literature. It may be difficult to duplicate published results because of the need to use a different supplier of TLC plates and densltometer scanner, as well as different visualization techniques. A method is described for using High Efficiency TiC plates (Analteoh HEq%C-HL) with an adjusted Chtoroform/(Methanol)/N}[~0H developing solvent which gave good separation of all common amniotic fluid phospholipld components except phosphatidyl serine in 7.5 cms a n d a fifteen minute running time. Visualization was by fluorescence produced after spraying with 8-anllino-1naphthalene sulfonic acid (ANSA) (Heyneman, R.A. et al (1972) d. Chromatogr. 68, 285). The increased sensitivity of this reagent versus common charring methods iS an advantage for measurement of the low phosphatidy] glycerol concentrations of interest. Pre-treatment of plates with cupric solution removes phosphatldyl serlne interference as It renders It non-fluorescent with the ANSA spray. Estimation can he made by visual comparison with standards or by scanning plates on Beckman Model CDS-200 Computing Densitometer System in fluorescence mode. Results are presented for linearlty of fluorescent response for phosphatidyl glycerol, lecithin, sphingomyeIin, and disaturated phosphatidyl choline in the range of clinical interest and for some typical amniotic fluids.
dard [2-(4-benzoylphenyl)-butyric acid, L.~.], a double extraction procedure is performed. The first step involves acidification with 0.5 ,~L of HCI I mol/L and extraction in an organic phase (9.5 mL of an hexane/iso-amyl alcohol mixture, 99/I). l, the second step, drug and I.S. are extracted from the organic phase with 1.0 ~uL of a phosphate buffer, 30 ~ o l / L , pH 8.0. We inject 250 uL of the aquous phase. The reverse phase chromatographic conditions are: stationary phase, C18 bonded phase; mobile phase, 50% acetonltrile, 50% phosphate buffer 45 mmol/L, pH 3.0; flow rate, 2.0 mL/mln. The detector is set at a wavelength of 254 nm and at 0.05 absorbance units full scale. Average retention times are 5.9 min for l.S. and 8.8 min for indomethacin. In serum, no peak was found to interfere with indomethacln. Linearity was observed up to 5 mg/L. The lower limit of detection was 0.3 mg/L but, if needed, it could be easily lowered to 0.05 mg/L. The results of precision and accuracy studies are presented in the following table: Drug added (m$/L)
Within assay mean (m~/L)
0.7 1.5 3.0
0.71 1.53 3.06
PRECISION C.V. Between assay (%) mean (mB/L~
C.V. (%)
ACCURACY % Recovery ± i S.D.
4.3 1.6 1.8
7.3 6.8 6.1
98.9t 7.2 I01.2± 6.9 101.0± 6.21
0.69 1.52 3.03
Preliminary data indicate tha~ other anti-inflammatory drugs (including ¢arprophen, ibuprophen, naproxen and ketoptophen) could be measured with this same method (or minor modifications).
2~ IDENTIFICATION AND ANALYSIS OF TRIS(-BUTOXYET}~L) PHOSPHATE BY A DUAL-COLUMN AND DUAL-DETECTOR CLC PROCEDURE Suria~ D. and Wright, L.A. Dept. Clin. Biochem. The Wessesley Hospital, Toronto, Ontario M4Y 133 Trls(-butoxyethyl)phosphate (TBEP), a constituent of stoppers in certain blood collecting tubes, has been shown to lower the plasma level of some basic drugs spuriously (gorge. O. et.al. (|977) Clin. Pharmacol, Therap. 22:539). Until these stoppers can be completely eliminated an attempt has been made to identify TBEP in the blood sample or stopper and to assess its possible laterference with the analysis of drugs by means of gas chromatography (GO). A Hewlett-Packard 5880 GC equipped with a 3.8% SE-30 column/ FID detector and a 3% SP-2250 column/NP detector was used. The analyses were performed simultaneously on both columns at [sothermlc oven temperature set at 250 C. TBEP (Aldrich 13,059-]) was detected as a single peak on the SE-30 system at a retention time (RT) Of approximately 2.5 min, and as three peaks on the SP-2250 column system at RT 0.6, 1.4 and 4.5 min. TBEP has been found to cause a shift of disopytamide, quinidine, lidoealne, procainamide and propranolol into the erythrocyte, thus lowering the plasma level. In add|flea, it will interfere with drugs having the retention times in the region of those given above.
21
THE USE OF SILICA SEP-PAE C A / ~ I D ~ FOR PURIFICATION OF CHOLESTERYL ESTERS FR~I HUMAN SERiaL WANG, S.T., and Peter, F., Biochemistry Laboratory, Laboratory Services Branch, Ontario Ministry of l)ealth, Toronto, Ontario M~q 5K9. A simple and fast analytical procedure for separation and purification of cholesteryl esters of human serum is described: A single lipid extract, together with cholesteryl pentadecanoate, as an internal standard, was passed through a silica SEP-PAK (Waters Scientific) and was eluted with 1.5% ethyl ether in petroleum ether. With this solvent system, only cholesteryl esters were eluted from the coltm~. The separation was verified with TIC system On silica gel with petroleum ether/e~]yl ether/glacial acetic acid (80/20/]) as a solvent. It showed a very clean TLC chromatogram of cholesteryl esters without any additional spots. The cbolesteryl esters were quantified by analyzing their fatty acid c c ~ s i t i o n as methyl esters by GLC. The reprOducibility of the methOd was estimated by carrying out six replicate analyses on one serum sample. The coefficient of variatic~ was 0.8-2.9% for the major fatty acids (C16:0, C18:1 and C18:2) and 2.0-]6.6% for the minor fatty acids examined, (C16:1, C18:0, C20:0 and C20:4). The recovery was determined by adding a known amount of cholesteryl ester standard of cholesteryl palm|tare, cholesteryl oleate and cholesteryl linofeats to a serum s ~ o l e , then assaying the sample with the method. The percent recoveries for cholesteryl palm|rate, cholesteryl oleate and cholesteryl linoleate were 90.7, 92.3 and 91.0% respectively. We have evaluated the usefulness of the SEP-PAK cartridges method over ~ e conventional TIC method for the clean up of the lipid extract of serum samples for the analysis of cholesteryl esters. The method is suitable for the measurement of both cholesteryl esters of the total and HDL fraction in small serum sanples.
22
SERUM INDOMETHACIN DETER}IINATION BY HIGH PRESSURE LIQUID CHROMATOGRAPHY. Leclercr P., Billon, B., and PetitClerc, C., Centre Hospitaliet Universitalre de Sberbrooke, Sherbrooke, Quebec JIH 5N4 We present and evaluate a HPLC method for the measurement of Isdomethacin in serum. After adding 2 mg/L of the internal stan-
2q ADAPTATION OF THE BMC ENZYMATIC CREATININE TO THE ILMULTISTAT III: CLINICAL EVALUATION. Sold|n, S.J. and McPherson, K.A., Dept. Clin. Biochem. and Pharmacol., Univ. of q~Dronto, and Res. Inst., Heap. for Sick Children, Toronto, Ontario MbG IX8. The enzymatic treat|nine procedure employing treat|nine amidohydrolase has been well documented in the literature (Wahlefeld, A.W. et el. (1972) Scand. J. Clin. Lab. Invest. 29, Suppl. 126, Abstract ,~30.i and Jaynes, P. et al. (1981) Clin. Chem. 27, 1034, Abstract ~052). We have adapted the Boehringer Mannheim p r O C e dure to the IL-Multistat llI. blultistat settings used were: T=30°C, ~=340 am, delay = 3s, interval = 540s. Loader settings were: sample volume 20 ul (40 ul flush), reagent volume I00 lJl (10 lJl flush), blank switch on diluent. The reagent mix used was 4.95 ml reagent i, 0.15 ml reagent 2, 0.30 ml reagent 3, and either 75 ul of reagent 4 or 75 ul of water. (The latter is used for the blank run). Results for standards, controls and samples are calculated by plotting &A (=AAcreatininase - ~Awater - AAreagent blank) vs. creatinine concentration. The between-day precision at serum concentrations of 4.2, 8.4 and 28.9 mg/L qave coefficients of variation of 14.1%, 7.2% and 2.5% respectively (n=2O). Addition of 20, 40 and 66.6 mg/L oF creatinlne to a plasma pool resulted in recoveries of 100%, 99% and 95% respectively (n=lO) . Patient Population n ~ ~ r Slope Intercept BbIC Beckman Premature and fullterm infants 72 0.65 0.80 O.9199 1.162 O.04R Nephrology excluding dialysis patients 69 2.28 2.37 0.9859 1.056 0.040 Dialysis patients 20 7.66 8.54 0.9536 0.8964 1.673 The 95th percentile for the premature and full-term infants was found to be 10.6 mg/L.