Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs. Application to tocainide

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride-sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol-phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 microgram/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 microgram/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.     

Determination of streptomycin in serum by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for monitoring the serum concentration of streptomycin. The method includes clean-up using a Sep-Pak C18 cartridge and quantitation using dihydrostreptomycin as an internal standard. Streptomycin and dihydrostreptomycin were separated by reversed-phase ion-pair chromatography on LiChrosorb RP-18 and detected by UV absorption (195 nm). The calibration graph of serum streptomycin concentration was linear over the range 5-50 micrograms/ml. Streptomycin was added to serum at the level of 20.0 micrograms/ml and its concentration was determined to be 18.9 micrograms/ml with a coefficient of variation of 2.07% (n = 5). The clinical application of this method was confirmed by comparison with fluorescence polarization immunoassay.     

Determination of malotilate and its metabolites in plasma and urine

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.     

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).     

New simplified microassay for the quantitation of theophylline and its major metabolites in serum by high-performance liquid chromatography

A high-performance liquid chromatographic technique is presented for simultaneous determination of theophylline, 3-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid and caffeine in serum using beta-hydroxyethyltheophylline (BHET) as an internal standard. An aliquot of a serum sample (100 microliters) was added to 40 microliters of an internal standard solution (BHET; 100 micrograms/ml) and vortexed. A 20% trichloroacetic acid solution (60 microliters) was then added; the mixture was vortexed, centrifuged and 100 microliters were injected onto a C8 column maintained at 45 degrees C. Inter- and intra-day variability of the assay for all compounds was less than 4.3%. Sensitivity ranged from 10 ng/ml for 1-methyluric acid to 25 ng/ml for theophylline. Drugs commonly coadministered with theophylline did not interfere with the assay.     

Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).     

Liquid chromatographic analysis of mexiletine in serum, with alternate application to tocainide, procainamide, and N-acetylprocainamide.

A simple and precise high performance liquid chromatographic method for the determination of mexiletine in human serum or plasma is described. Following addition of N-propionylprocainamide as internal standard the specimens are extracted, under basic conditions, into methylene chloride. After removal of the aqueous layer the drug is back-extracted into dilute acid, which is then injected directly for analysis. The extraction efficiency is 79% for both mexiletine and internal standard, and the assay is linear to 4 mg/L (twice upper therapeutic concentration). Inter-run coefficients of variation are 3.0% or less. The relative retention time of mexiletine to internal standard averages 1.3. An adaptation of this method is described for an alternate application to the analysis of tocainide, procainamide and N-acetylprocainamide.     

Determination of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid and related bile acids in human serum by gas chromatography-mass spectrometry

The glycine, taurine and sulphate conjugates of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid were synthesized as authentic samples for the analysis of this unusual bile acid. A highly sensitive and specific quantitative assay of the bile acid and related compounds has been developed by selected-ion monitoring in gas chromatography-mass spectrometry of their methyl ester-trimethylsilyl ether derivatives, using the deuterium labelled internal standards: [2H6]3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, [2H5]3 beta-hydroxy-5-cholen-24-oic acid, [2H7]cholic acid and their sulphates. Calibration curves for these bile acids were linear over the range 0.01-100 micrograms/ml in human serum. Recoveries of the bile acids and their conjugates ranged from 95 to 103% of the added amounts of their standard samples. The unsaturated bile acid was identified in a significant amount of 25.2 micrograms/ml in serum of an infant with liver disease, and its sulphate comprised 55.1% of the amount of the bile acid.     

Simultaneous determination of clarithromycin and 14(R)-hydroxyclarithromycin in plasma and urine using high-performance liquid chromatography with electrochemical detection.

A sensitive method for the simultaneous high-performance liquid chromatographic determination of clarithromycin and its active metabolite in plasma and urine is described. Alkalinized samples were coextracted with an internal standard and analyzed on a C8 column using electrochemical detection. Recoveries were greater than or equal to 85% and consistent. Standard curves for plasma were linear in the range 0-2 micrograms/ml for both compounds (r greater than 0.99), with limits of quantification of approximately 10.03 micrograms/ml (0.5-ml sample). Within-day and day-to-day precision were good, with coefficients of variation mostly within +/- 5%; accuracy for both compounds were routinely within 90-110% of theoretical values. Standard curves for urine were linear in the range 0-100 micrograms/ml with limits of quantification of 0.5 micrograms/ml (0.2-ml sample). Urine assays also had similar within-day and day-to-day precisions and accuracy.     

High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C18 column (Spherisorb, 5 micron) using a mobile phase of 2% glacial acetic acid, 30--35% acetonitrile, 70--65% water with ultraviolet detection (254 nm). The method uses 17 alpha-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 microgram/ml for HC, 0.07 microgram/ml for MP, 0.04 microgram/ml for MPHS, and 0.10 microgram/ml for HCHS, with a detection limit of 0.02 microgram/ml for all four steroids. Calibration curves are linear up to 3 micrograms/ml for MP or MPHS (as equivalent MP) and up to 4 micrograms/ml for HC and 7 micrograms/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate for each steroid is less than 7%. Plasma concentration--time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.     

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.     

Rapid and sensitive determination of aprindine in serum by gas chromatography using a surface ionization detector

A rapid and highly sensitive method for the determination in serum of aprindine, an antiarrhythmic drug, was developed employing gas chromatography with a surface ionization detector. No interfering peak from endogenous substances appeared when an organic phase was directly injected into the system after single extraction from a serum sample. A standard curve obtained was linear up to the serum level of 6 micrograms/ml, and the limit of sensitivity was 16 pg. The method described is applicable to routine therapeutic monitoring of serum concentrations of aprindine.     

Plasma determination of the novel anticonvulsant D,L-3-hydroxy-3-ethyl-3-phenylpropionamide and preliminary pharmacokinetic studies in the rat.

A sensitive gas chromatographic method with flame ionization detection was developed for the analysis in plasma of the novel anticonvulsant D,L-3-hydroxy-3-ethyl-3-phenylpropionamide (HEPP), using D,L-2-hydroxy-2-ethyl-2-phenylacetamide as the internal standard. HEPP was extracted from alkalinized plasma into dichloromethane and quantified after derivatization with bis(trimethylsilyl)-trifluoroacetamide: Standard curves were linear from 0.5 to 50 and from 2 to 100 micrograms/ml of plasma, using 1.5 and 5 micrograms of the internal standard, respectively. The lower limit of detection at a signal-to-noise ratio of 3 standard deviations was 0.33 micrograms/ml of sample. The sensitivity, accuracy and reproducibility of the method were shown to be satisfactory for pharmacokinetic studies of HEPP. After intraperitoneal administration of 50 mg/kg to Wistar rats, the principal kinetic parameters were: absorption half-life = 0.04 h; volume of distribution = 1.32 l/kg; clearance = 4.40 ml/min; peak concentration = 50 micrograms/ml; peak time = 0.25 h; mean residence time = 4.55 h.     

Analytical procedure for the determination of rufloxacin, a new pyridobenzothiazine, in human serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantification of rufloxacin in human serum and urine has been developed and validated. The compounds, rufloxacin and internal standard, are extracted from buffered serum and urine using dichloromethane. They are then separated on an anion-exchange column using 0.05 M phosphate buffer-acetonitrile (80:20, v/v). The eluate is quantified by measuring the ultraviolet absorbance at 296 nm. The lower limit of detection for the analyte is 0.1 microgram/ml in serum and 0.05 micrograms/ml in urine. The method is linear from 0.3 to 10 micrograms/ml for serum and 0.1 to 10 micrograms/ml for urine. The method has been applied in a pharmacokinetic study in volunteers.     

Sensitive method for the determination of the antiarrhythmic drug detajmium in serum by solid-phase extraction and high-performance liquid chromatography with fluorimetric detection.

The objective of this study was to develop a very sensitive and selective method for the determination of detajmium (4-3-diethylamino-2-hydroxypropyl-ajmaline), a sodium-channel-blocking drug with antiarrhythmic properties, in serum. A high-performance liquid chromatography (HPLC) method with solid-phase extraction and fluorimetric detection has been applied. Serum samples were diluted with phosphate buffer (pH 3.5) and the extraction of detajmium and ajmaline, which was used as an internal standard, was carried out with Oasis cartridges (Waters). The chromatographic separation was performed on a RP18 column. The limit of quantification for serum samples of detajmium was 1 ng/ml with good reproducibility (R.S.D. < 15%) and a linear response from 1 to 200 ng/ml. The described method is highly sensitive and specific for the determination of detajmium in serum of patients and volunteers.     

Simultaneous separation and determination (in serum) of phenytoin and carbamazepine and their deuterated analogues by high-performance liquid chromatography--ultraviolet detection for tracer studies

The use of stable isotope-labeled tracer compounds is the safest and most effective method to perform many steady state pharmacokinetic and drug interaction studies. We describe a method by which the heavily deuterated 2H10 analogues of carbamazepine (2H10 CBZ) and phenytoin (2H10 PHT) can be chromatographically separated by high-performance liquid chromatography from unlabeled CBZ and PHT. All compounds are quantitated against an internal standard (IS) (10,11-dihydrocarbamazepine) and measured using conventional UV detection rather than mass spectrometry. Baseline resolution of extracted serum containing 2H10 CBZ, CBZ, 2H10 PHT, PHT and IS is achieved on a heated (55 degrees C) 25 cm x 4.6 mm BioAnalytical Systems Phase II 5 microns ODS column with an isocratic mobile phase consisting of water-acetonitrile-tetrahydrofuran (80:16:4, v/v/v) at 1.2 ml/min. Eluting compounds were monitored at a UV wavelength of 214 nm. Calculated resolution of 2H10 CBZ from CBZ and of 2H10 PHT from PHT were 1.3. Serum standard curves were linear (R greater than or equal to 0.999) over a range of 0.5-14 micrograms/ml for 2H10 CBZ, 0.5-20 micrograms/ml for CBZ, 0.5-20 micrograms/ml for 2H10 PHT, and 0.5-30 micrograms/ml for PHT. Within-day percent relative standard deviations (precision) were less than 6% in all cases.     

High pressure liquid chromatographic assay for mexiletine in serum

A highly sensitive, rapid, and specific high pressure liquid chromatographic assay for the analysis of the antiarrhythmic agent mexiletine is reported. The method involves extraction of mexiletine with organic solvent followed by analysis using fluorescence detection. The minimum measurable limit is 1 ng and inter- and intra-day coefficients of variation are less than 5.8%. The method is useful for pharmacokinetic studies and routine serum monitoring of mexiletine.     

Determination and Pharmacokinetics of Mexiletine in Rabbit Plasma by Gas Chromatography with Mass Spectrometry

This paper describes a gas chromatography/mass spectrophotometry method for determination of mexiletine in rabbit plasma. Mexiletine and internal standard metoprolol were extracted from rabbit plasma with a mixture of ethylacetate and diethylether at basic pH with liquid-liquid extraction. Calibration curves were linear over the concentration range 45–2000 ng/mL. Intra- and inter-day precision (relative standard deviation) for mexiletine in rabbit plasma were less than 6.9%, and accuracy (relative error) was better than 6.8%. Recovery of mexiletine from rabbit plasma averaged 92.6%. This method was successfully applied to six rabbits which had given an oral capsule of 200 mg mexiletine.     

High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues.

High-performance liquid chromatographic methods using reversed-phase chromatography and electrochemical detection have been developed for the quantitation of azithromycin in serum and tissues of laboratory animals and humans. Serum sample preparation involved addition of internal standard, alkalinization, and solvent extraction. Tissue sample preparation involved Polytron homogenization in acetonitrile containing internal standard, evaporation of the supernatant, alkalinization of the residue, and solvent extraction. Serum samples were chromatographed on an alkylphenyl-bonded silica column eluted with pH 6.8-7.2 mobile phase with a dual-electrode coulometric detector operated in the oxidative screen mode. Serum and tissue samples were chromatographed on a gamma RP-1 alumina column with pH 11 mobile phase with a glassy carbon amperometric detector. Recovery of azithromycin was 87% from serum and 85% from tissues. Linear standard curves were prepared in serum over two concentration ranges (0.01-0.20 and 0.20-2.0 micrograms/ml) and in tissues over several concentration ranges (0.1-2, 1-10, 10-100, and 100-1000 micrograms/g). In serum and tissues, intra- and inter-assay precision ranged from 1 to 8% and 4 to 11%, respectively. The tissue assay has been applied to liver, kidney, lung, spleen, muscle, fat, brain, tonsil, lymph nodes, eye, prostate and other urological tissues, and gynecological tissues.     

Selective liquid chromatographic assay for propylthiouracil in plasma

A liquid chromatographic assay was developed to quantitate propylthiouracil in plasma using an internal standard, 5-propyl-2-thiouracil, of similar structure and physical properties. Caffeine, which coelutes with propylthiouracil, was removed by extraction from serum treated with base. No other compounds were found to interfere in the assay. The drug was extracted from plasma with chloroform with a recovery of 59.4% and the intra- and inter-assay coefficients of variation were 5.7 and 3.3%, respectively. The assay was linear to 3 micrograms/ml with a lower detection limit of 40 ng/ml for a sample volume of 1 ml.