A New Simplified Microassay for the Quantitation of Theophylline in Serum by High-Performance Liquid Chromatography

Abstract

An improved, rapid and simplified HPLC method is presented for the quantitation of theophylline in serum. The internal standard, beta-hydroxypropyl theophylline (20 ul of a 100 ug/ml solution) is added to 50 ul of serum. Serum proteins are precipitated by the addition of 30 ul of 20% trichloroacetic acid solution. The mobile phase consists of a sodium acetate buffer with 8% acetonitrile. Chromatogram run time is 8 minutes. The sensitivity limit is 0.10 ug/ml. This method is interference free from the major metabolites of theophylline and other drugs commonly coadministered with theophylline.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.     

Sensitive determination of piritramide in human plasma by gas chromatography.

A selective and sensitive method for the determination of piritramide in human plasma is described. A 1-ml aliquot of plasma was extracted with 10 ml of hexane-isoamyl alcohol (99.5:0.5, v/v) (extraction efficiency 86%) after addition of 50 microliters of 2 M ammonia and 20 microliters of aqueous strychnine solution (100 ng per 10 microliters) as internal standard. Gas chromatography was performed with J&W DB-1, 30 m x 0.53 mm I.D. separation column, film thickness 1.5 microns, using an nitrogen-phosphorus-sensitive detector. The assay was linear in the concentration range 3.75-2250 ng/ml (r = 0.999), with a lower limit of detection of 1-2 ng/ml. The precision was determined using spiked plasma samples (10 and 50 ng/ml), with coefficients of variation of 3.5 and 3.1% (intra-day; n = 5) and 4.6 and 4.1% (inter-day; n = 4). In the range 3.75-150 ng/ml, the accuracy of the assay was 3.36%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or post-operative analgesia.     

An HPLC Method for the Determination of Theophylline and Its Metabolites in Serum and Urine

Abstract

A urine and a serum assay have been developed to quantitate theophylline and its major metabolites:1,3-dimethyluric acid, 3-methylxanthine and 1-methyluric acid. Reverse phase chromatography follows a serum acetone extraction procedure and a urine anion exchange clean-up procedure. Lower limits of sensitivity are 0.04 μg/ml for serum metabolites and 1 μg/ml for urine metabolites. Both assays are free of interference from endogenous substances. These assays have been tested successfully in pharmacokinetic and metabolic studies of theophylline.     

Extractionless determination of diclofenac sodium in serum using reversed-phase high-performance liquid chromatography with fluorimetric detection.

The author describes a method of using reversed-phase high-performance liquid chromatography with fluorimetric detection for the assay of diclofenac sodium in serum. The method is sensitive down to 20 ng/ml (250-microliters loop). Elution is at pH 6.2 with methanol in 0.05 M phosphate buffer (43:57, v/v) on a 25-cm Spherisorb S5 ODS2 column. Detection is at an excitation wavelength of 282 nm and an emission wavelength of 365 nm. Serum sample size is 100 microliters. Sample protein, to which diclofenac is highly bound, is first denatured by heat and then with methanol to release the diclofenac prior to centrifugation and injection of 100 microliters (or 250 microliters) of the clear supernatant. Harmol, with similar fluorescence and polarity characteristics to diclofenac, is a satisfactory internal standard. At the 1 micrograms/ml level intra-sample reproducibility is better than 2%, whilst inter-sample reproducibility is 4.6%. Detector response is linear from 40 ng/ml to 20 micrograms/ml (100-microliters loop).     

A method for the determination of theophylline in serum by isotope dilution mass spectrometry

Summary A method for the determination of theophylline in human serum by the isotope dilution/mass spectrometric technique is described. As an internal standard labelled (1,3-¹⁵N₂-2-¹³C)theophylline is added to the serum sample. The analyte and internal standard are extracted with chloroform/2-propanol (90:10) and converted to the trimethylsilyl derivatives. The extraction and silylation procedures are checked by adding theophylline and internal standard in various concentrations to blank serum and determining the recovery. The trimethylsilyl derivatives of labelled and non-labelled theophylline are separated and detected by GC-MS with the mass spectrometer set to m/z 252 and 255. The amounts of theophylline in the serum are calculated from the isotope ratios measured by selected ion monitoring. The accuracy, precision and recovery of this GC-MS method are presented and discussed. The coefficient of variation determined from duplicate samples was less than 2.5%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1.Part of the work was presented at the 32th Kongreß der Deutschen Gesellschaft für Laboratoriumsmedizin, Frankfurt 1991     

Fluorimetric determination of mexiletine in serum by high-performance liquid chromatography using pre-column derivatization with fluorescamine

A simple, specific and sensitive micro-scale method for the assay of the antiarrhythmic agent mexiletine in human serum is described. The method uses high-performance liquid chromatography, with pre-column fluorimetric derivatization by fluorescamine. Following extraction with diethyl ether, mexiletine and 4-methylmexiletine (an internal standard) were derivatized with fluorescamine under weakly alkaline condition (pH 9.0) and chromatographed on a reversed-phase column with aqueous methanol-2-propanol as the mobile phase. The two fluorescent derivatives of mexiletine and the internal standard were separated as clear single peaks, and no interfering peaks were observed on the chromatograms. The detection limit for mexiletine was 0.005 micrograms/ml from only 100 microliters of serum, and the calibration curves in the range 0.01-5 micrograms/ml were linear, with an overall coefficient of variation of less than 5%. The analytical recovery of a known amount of mexiletine added to serum was almost 100%. This method proved to be effective in the rapid monitoring of the serum concentrations in patients who received this potent antiarrhythmic drug.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Simultaneous determination of methylprednisolone and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt in human urine by high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 243 nm has been developed for the quantitative determination of methylprednisolone (MP) and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt (MPSO) in human urine following therapeutic doses in humans. The assay procedure involves stabilization of urine samples by addition of disodium ethylenediaminetetraacetic acid (Na2EDTA) and ion-pair extractions of MPSO using tetraethylammonium chloride (TEACl) as the counter ion. After extracting both drugs and internal standard into chloroform, the extract was evaporated to dryness under nitrogen. The resulting residue was reconstituted in 200-500 microliters of mobile phase and chromatographed on an IBM C18 reversed-phase column (5 microns). The mobile phase was a mixture of water-acetonitrile-isopropanol (71.2:18.8:10.0, v/v) containing 75 microliters of 0.1 M hydrochloric acid and 0.450 g of TEACl per liter. Propyl p-hydroxybenzoate was used as an internal standard. The extraction efficiencies of MP and MPSO were greater than 90% using the ion-pairing agent TEACl. The chromatographic responses were linear up to about 200 micrograms/ml for MP and 80 micrograms/ml for MPSO and had sufficient precision and accuracy to provide quantitative data from human urine. The assay detection limit was about 8 ng/ml for MP and 25 ng/ml for MPSO in human urine. Stability studies in urine indicated that without Na2EDTA stabilization and at room temperature, rapid degradation of MPSO occurred in urine. Addition of EDTA to the urine specimen and storage at -70 degrees C increased the stability of MPSO, and little or no degradation was observed in urine stored for more than 60 days. The method has been used in the simultaneous determination of MP and MPSO in urine specimens obtained from a single-dose tolerance study of MPSO in normal male volunteers.     

Automated microanalysis of carbamazepine and its epoxide and trans-diol metabolites in plasma by column liquid chromatography.

A fully automated high-performance liquid chromatographic procedure for the simultaneous determination of carbamazepine and its main metabolites, epoxycarbamazepine and dihydroxycarbamazepine, in plasma is described. Liquid-solid extraction on disposable C18 columns and reversed-phase chromatography on a 3 microns particle size C18 column were combined and automated by using the Automatic Sample Preparation with Extraction Columns system. Ultraviolet detection was performed at 210 nm. 5,6-Dihydro-11-oxo-11H-dibenz[b,e]azepine-5-carboxamide was used as internal standard. A small plasma volume (100 microliters) was required. The total run time for the assay of one sample was about 10 min. The assay demonstrated good reproducibility. The limit of quantitation was 0.1 mumol/l (about 25 ng/ml).     

Simplified procedure for the determination of sotalol in plasma by high-performance liquid chromatography

A simple, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) procedure for sotalol determination is described requiring small plasma volumes. The high recovery of sotalol from plasma and the high precision of measurement obviate the need for an internal standard. Plasma samples (300 microliters) were deproteinised with 50 microliters of 70% (w/w) perchloric acid in disposable glass tubes. After vortex-mixing and centrifugation, 30 microliters of 4 M K2HPO4 were added followed by gentle shaking. A 20-microliters aliquot was then injected (by autosampler) for HPLC analysis. Chromatography was performed on a glass-lined 250 mm x 4 mm 5-micron C18 steel column. The mobile phase was 6% (v/v) acetonitrile in 0.08 M KH2PO4 buffer (pH 4.6). The flow-rate was 0.8 ml/min. Detection was by fluorescence with excitation and emission wavelengths at 235 and 310 nm, respectively. The retention time for sotalol was 7.1 min. Calibration was linear from 0.16 to 10 micrograms/ml in plasma (r greater than 0.999 for detector response to sotalol). The minimum concentration for quantitation was 0.08 micrograms/ml [within assay coefficient of variation (C.V.) less than 5%]. Recovery was near quantitative (greater than 98%) and replicate (intra-assay precision was less than 5% C.V.). Analysis of samples (n = 10) at concentrations of 0.42 and 4.2 micrograms/ml gave mean values of 0.44 and 4.3 micrograms/ml, respectively. The inter-assay C.V. values were 4.5 and 2.2%, respectively. Other clinically used antiarrhythmic drugs did not interfere. This assay can be performed using other commercial C18 analytical columns by suitable adjustment of mobile phase flow-rate and acetonitrile composition.     

Liquid chromatographic analysis of clonazepam in human serum with solid-phase (Bond-Elut) extraction

A simple, sensitive, selective and precise liquid-column chromatographic assay for clonazepam is described, in which 1 ml of serum containing 50 micrograms/l methylclonazepam as an internal standard is extracted by elution from a Bond-Elut column with 400 microliter of methanol. An aliquot of the eluate is injected on to a reversed-phase column and eluted with a mobile phase of acetonitrile--phosphate buffer (30:70) at a flow-rate of 2 ml/min at a column temperature of 50 degrees C. Detection is at 254 nm. Chromatography is complete in 12 min. A sensitivity of 2 ng/ml is attained when 1 ml of serum is extracted. Analytical recovery of the clonazepam added to serum ranged from 91% to 99% with a coefficient of variation of 6.0%. This assay for clonazepam has good precision, with coefficients of variation of 11% at 15 ng/ml and 2.6% at 50 ng/ml. There was no interference from any of the commonly used antiepileptics.     

Determination of sorbic acid in urine by gas chromatography-mass spectrometry.

The average daily uptake of the common food preservative sorbic acid is estimated to range from 0.01 to 1.1 mg kg-1. Sorbic acid mainly is metabolised to carbon dioxide. Minor amounts are converted to trans,trans-muconic acid (ttMA) as well as excreted unchanged into the urine. Since urinary ttMA is a biomarker for the occupational and environmental exposure to benzene, there is an additional need for monitoring the uptake of sorbic acid, particularly at low, environmental benzene exposure levels. For this purpose, a simple, robust and rapid method for the determination of sorbic acid in urine at trace levels was developed. After addition of 10 ml of water and 5 ml of 8 M hydrochloric acid to 10 ml of the thawed urine, the sample was water steam distilled using an automated distillation device. A total of 100 ml of the distillate were solid-phase extracted. After washing, the sorbic acid was eluted with 4 ml methanol. The eluate was reduced under a stream of nitrogen to a volume of 300 microliters. After addition of 500 microliters boron trifluoride in methanol and incubation for 1 h at 60 degrees C, the resulting sorbic acid methyl ester was extracted three times with 1 ml heptane. To the combined heptane layers, sorbic acid ethyl ester was added as an internal standard. After reducing to a volume of 100 microliters in a stream of nitrogen, the final analysis was performed by GC-MS using the fragment ions m/z 126 for the analyte and m/z 140 for the internal standard. The limit of detection was 0.7 ng ml-1 urine and the R.S.D. of 69 duplicate determinations was 7.5%. In a controlled, experimental study and in a field study, we were able to show that urinary sorbic acid is a marker for the dietary uptake of sorbic acid and that sorbic acid is converted to ttMA. On average, 0.1% of the dietary sorbic acid is excreted unchanged into the urine. Excretion is complete within 24 h. We found that, on average, 0.23% of the oral dose of sorbic acid is excreted as urinary ttMA. There was a significant correlation between urinary excretions of sorbic acid and ttMA (r = 0.74, n = 69). We conclude that urinary sorbic acid can be used to correct the urinary ttMA level in order to determine the portion related to benzene exposure. This appears to be necessary particularly at low, environmental benzene levels.     

Measurement of penbutolol and 4-hydroxypenbutolol in plasma or serum by HPLC

A simple HPLC method for penbutolol and 4-hydroxypenbutolol assay has been developed. Plasma or serum (200 microliters) is vortex-mixed (30 s) with Tris solution (2 M, pH 10.6) containing an internal standard (50 microliters) and methyl t-butyl ether (200 microliters). After centrifugation, the extract (100 microliters) is analysed using an unmodified silica column (250 x 5 mm ID) and iso-octane-methanol-methyl t-butyl ether (55:25:20) containing ammonium perchlorate (10 mM, pH 5.7) as eluent and with fluorescence detection. No interference has been encountered and the limit of accurate measurement for both compounds is 5 micrograms/l.     

Determination of telenzepine in human serum by gas chromatography-mass spectrometry

A method for determining telenzepine in human serum is described. Analytes are obtained from alkalinized serum by extraction of the drug using reversed-phase octadecylsilane-bonded silica cartridges. Telenzepine and a desmethyl analogue added to serum as internal standard are retained on the C18 cartridge and recovered by elution with methanol. The gas chromatographic properties of telenzepine and the internal standard are improved by a two-step derivatization involving a benzodiazepinone-benzimidazole rearrangement and simultaneous formation of a methyl ester function. The processed extract is analysed by gas chromatography-mass spectrometry with selected-ion monitoring. Quantification is linear over the range 2-40 ng/ml. Inter-day precision is within 7%, except at the detection limit of 2 ng/ml (16%). Application of this assay to routine analysis is limited by the extensive sample pretreatment essential for derivatization of telenzepine.     

Determination of nicotinic acid in serum by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of nicotinic acid in serum is described which employs high-performance liquid chromatography with fluorescence detection. Nicotinic acid and 2-chloronicotinic acid as an internal standard in deproteinized serum are reacted with N,N'-dicyclohexyl-O-(7-methoxycoumarin-4-yl)methylisourea in acetone to give the corresponding fluorescent 4-hydroxymethyl-7-methoxycoumarin esters. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18 with isocratic elution using aqueous acetonitrile containing a small amount of sodium 1-hexanesulphonate as a mobile phase. The detection limit of nicotinic acid in serum was 0.2 nmol/ml. The method requires only 100 microliters of serum.     

A gas chromatographic method for measuring 6-mercaptopurine in serum.

A method is presented for the measurement of 6-mercaptopurine (6-MP) in serum. Serum samples containing the drug were treated with dithioerythritol and saturated with ammonium carbonate. 6-MP was then extracted from the serum with a mixture of isopropanol-ethyl acetate (1:1, v/v) containing 0.01% ethanethiol. The solvent was evaporated, the residue dissolved in dilute hydrocholoric acid, washed with chloroform, and extracted with ethyl acetate. 6-MP was derivatized with trimethylanilinium hydroxide and measured gas chromatographically. The sulfhydryl-protecting reagents, dithioerythritol and ethanethiol, were added to prevent the decomposition of 6-MP. The extraction and clean-up method recovered 78 +/- 2% (S.E.) of the 6-MP present. Tracer amounts of [8-14C]6-MP served as the internal standard during the extraction part of the method. Theophyline was used as the internal standard during the gas chromatographic analysis. The standard curve obtained from the gas chromatograph was linear between 0.5 and 20 mug/ml of 6-MP. Serum samples were stored in the freezer for two weeks without significant loss of drug. No interference was encountered from normal serum constituents or xanthines, such as caffeine or theophylline added to serum.     

A Micro Method for the Quantitation of Tryptophan in Biological Fluids by HPLC-EC

Abstract

A micromethod for the quantitation of tryptophan in biological fluids, such as whole blood, RBC, serum, plasma or tissue homogenates using HPLC and electrochemical detection is described. -Methyl tryptophan is used as an internal standard. Ten microliters of the sample are added to a mixture of 50 ul of acetonitrite and 10 ul of internal standard (20 ng/ul). This mixture is then diluted with 180 ul of triethylamine phosphate-buffer (pH 3.0). After centrifugation 25 ul of the sample are injected into HPLC system. Standards containing 100 ng and 200 ng of tryptophan are similarly prepared and 25 ul aliquot injected into HPLC.     

Liquid chromatographic assay for the simultaneous determination of pyrazinamide and rifampicin in serum samples from patients with tuberculous meningitis

A simple high-performance liquid chromatographic assay for the simultaneous determination of pyrazinamide and rifampicin in serum from patients with tuberculous meningitis is presented. The drugs and internal standard, p-acetamidobenzoic acid, were extracted from the acidified sample containing 2% ascorbic acid at pH 4.2 into dichloromethane-diethyl ether (2:3). The solvent extract was evaporated to dryness with the aid of nitrogen and the residue redissolved in methanol (75 microliters). The concentrate was analysed by a liquid chromatograph using a reversed-phase 30-microns C8 pre-column linked to a 5-microns C8 analytical column with a gradient solvent programme, which delivered 6% to 48% (v/v) acetonitrile in phosphate buffer (10 mM potassium dihydrogenphosphate, pH 3.5) in 10 min at 1.5 ml/min. The eluate was detected at 215 nm. Twelve patients with tuberculous meningitis were given daily chemotherapy, and their serum samples were assayed for pyrazinamide and rifampicin.