Rapid High-performance Liquid Chromatographic Assay Research Articles

Association of leukocyte telomere length with oxidative stress in yoga practitioners.

Yoga is a mind-body modulation technique that has been shown to have beneficial effects on various diseases related to various systems in the body. However, the molecular basis of mechanism of action is not clear. Hence, this study was designed to study the leukocyte telomere biology and its relation with homocysteine and oxidative stress in yoga practitioners. This is a prospective case-control study involving Yoga practitioners aged 30-40 y with minimum of two years yoga practice (Yoga group) and age, gender and body mass index matched sedentary healthy general population with no medical problems (Control group). Leukocyte telomere length (LTL) was measured by using quantitative PCR (qPCR), fasting plasma homocysteine was measured by a rapid high-performance liquid chromatography assay and the oxidative stress was assessed with total antioxidant status (TAOS), malondialdehyde (MDA) measured by calorimetry. The LTL was shorter in control group than in yoga group (p<0.001). The TAOS was more in yoga group when compared to control group (p=0.008), MDA and homocysteine was high in control group when compared to Yoga group (p<0.001). Further, the LTL was positively correlated with TAOS (r=0.841, p<0.001) and negatively correlated with MDA (r=-0.931, p<0.001) and Homocysteine (r=-0.756, p<0.001). The LTL is well-preserved in people who practice Yoga regularly with lower systemic oxidative stress compared to those who have a relatively sedentary lifestyle despite lack of any medical disorders. The habitual yoga practice seems to inhibit replicative cellular senescence.

HPLC-UVD를 이용한 축산식품 중 Novobiocin의 시험법 확립

노보비오신은 동물용의약품 중 약산성 항생제이며, 우리나라의 경우 선진국에 비해 항생제의 사용이 높은 만큼 동물용의약품의 잔류 사례는 계속해서 증가하고 있다. 이에 따라 식품 중 잔류동물용의약품의 안전관리를 위해 축산 식품 중 닭고기, 소고기 및 우유에서 노보비오신에 대한 HPLC-UVD를 이용한 잔류 분석법을 확립하였다. 각 시료 일정량에 메탄올을 가하고 균질화하여 대상 성분을 추출하고 액-액분배법으로 지방성분을 제거한다. 대상성분이 추출된 여액을 실리카 카트리지를 이용하여 정제한 다음 HPLC-UVD로 분석하였다. 확립된 시험법의 정량한계와 직선성은 CODEX 기준에 적합한 수준으로 각각 0.02 mg/kg과 0.999 (<TEX>$r^2$</TEX>)이었으며 회수율은 닭고기, 소고기 및 우유에서 각각 <TEX>$88.8{\pm}5.6-100.3{\pm}4.4$</TEX>, <TEX>$88.8{\pm}7.2-97.0{\pm}3.2$</TEX> 및 <TEX>$88.1{\pm}4.3-92.8{\pm}3.6%$</TEX>이었다. 본 연구에서 확립된 노보비오신의 시험법은 동물용의약품의 안전관리를 위한 공정시험법으로 활용될 것으로 기대된다. Novobiocin is a coumarin-containing antibiotic, and has a longer half-life in various animals than other veterinary medicines. A simple and rapid high-performance liquid chromatography assay for the determination of residual novobiocin levels in chicken, beef and milk has been developed and validated. The separation condition for HPLC/UVD was optimized by a MG II <TEX>$C_{18}$</TEX> (4.6 mm <TEX>$ID{\times}250$</TEX> mm, 5 <TEX>${\mu}m$</TEX>) column with 0.1% formic acid in <TEX>$H_2O$</TEX>/0.1% formic acid in Acetonitrile (40/60, v/v) as the mobile phase at a flow rate of 1.0 mL/min and the detection wavelength was set at 340 nm. Residues were extracted from tissue by blending with methanol. After liquid-liquid partitioning, lipid materials were removed with n-hexane and purification as Silica (1 g, 6 mL) cartridge with 10 mL acetone/dichloromethane (10/90, v/v). Limit of quantification and linearity performed by the analytical method were 0.02 mg/kg and 0.999 (<TEX>$r^2$</TEX>), and the recovery range was <TEX>$88.8{\pm}5.6-100.3{\pm}4.4$</TEX>, <TEX>$88.8{\pm}7.2-97.0{\pm}3.2$</TEX> and <TEX>$88.1{\pm}4.3-92.8{\pm}3.6%$</TEX>. It is expected that this analytical method with regards to novobiocin in chicken, beef and milk could be applied as an official method to administer food safety on veterinary medicines.

Development and Validation of a Rapid HPLC Assay for the Simultaneous Determination of Three Psychoanaleptic Drugs in Pharmaceutical Formulations

Atomoxetine, venlafaxine, and fluoxetine are classified as psychoanaleptics. While atomoxetine is used for the treatment of attention deficit/hyperactivity disorder (ADHD), the other two are for depressive disorders. A simple, rapid, isocratic, high performance liquid chromatographic (HPLC) method has been developed for the determination of atomoxetine, venlafaxine, and fluoxetine in pharmaceutical products. The HPLC method involves separation by reversed phase HPLC on a Nova‐Pak C18 column (15 cm×3.9 mm id) using acetonitrile‐potassium dihydrogen phosphate buffer (0.05 M adjusted to pH 3.0 with phosphoric acid) (45:55 v/v) as the mobile phase, at a flow rate of 0.8 mL/min at room temperature (25°C), using UV detection at 226 nm. The method was validated for specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and robustness. The excipients present in the formulations do not interfere with the assay procedure. The developed method was successfully applied to determine atomoxetine, venlafaxine, and fluoxetine in pharmaceutical formulations.

Optimization and validation of a dissolution test for selegiline hydrochloride tablets by a novel rapid HPLC assay using a monolithic stationary phase

The present study reports the optimization and validation of a dissolution test for selegiline·HCl tablets using a new high-performance liquid chromatographic (HPLC) method. Rapid separation of the analyte from sample matrix was achieved in less than 60 s using a Cromolith ® RP-18e monolithic column using UV detection at 220 nm. Thorough validation of the assay based on pre-defined criteria included linearity, LOD/LOQ, accuracy, precision, selectivity and ruggedness. The dissolution test was optimized in terms of dissolution medium, basket (type I)/paddle (type II) agitation and rotation speed. Its ruggedness was also validated. The presented analytical and dissolution procedures are currently being applied in the quality and stability control of Cosmopril ® tablets (5 mg/tablet selegiline·HCl, Cosmopharm Ltd., Korinthos, Greece).

Penetration of levofloxacin into the anterior chamber (aqueous humour) of the human eye after intravenous administration

In the study presented here, levofloxacin concentrations in serum samples and the aqueous humour (AH) of 16 patients undergoing cataract extraction were measured in order to determine the penetration characteristics of levofloxacin into the AH of the non-inflamed human eye. Cataract removal was performed at various times (from 90 to 270 min) after the end of a 30-min intravenous infusion of 500 mg of levofloxacin. Serum samples were obtained 1 h after the end of levofloxacin administration (Cmax); AH and a second serum sample were taken simultaneously during the operation, and the concentrations of levofloxacin in AH (C(AH)) and serum (C(S)) were determined using a rapid high-performance liquid chromatography assay. The mean Cmax was 6.07 microg/ml (range 3.75-9.53 microg/ml, SD 1.83). The mean C(AH) at the first hour following levofloxacin administration was 1.37 microg/ml (range 1.17-1.6 microg/ml, SD 0.22) and the mean ratio (R=C(AH)/C(S)) was 0.26 (range 0.24-0.3, SD 0.02). The mean C(AH) at 125-270 min following levofloxacin administration was 1.39 microg/ml (range 0.82-1.98 microg/ml, SD 0.33) and the mean R was 0.3 (range 0.15-0.53, SD 0.11). Of 16 patients, 15 had a C(AH) of >1 microg/ml 1 h after levofloxacin administration. In conclusion, 1 h after administration of 500 mg of levofloxacin, the levels obtained were higher than the MIC at which 90% of methicillin-susceptible Staphylococcus aureus and certain gram-negative bacteria strains are inhibited.

A specific and rapid HPLC assay for the determination of cefroxadine in human plasma and its application to pharmacokinetic study in Korean

A specific and rapid high performance liquid chromatographic (HPLC) method with UV detection (254 nm) was developed for the determination of cefroxadine in human plasma. The sample extraction was performed by a simple procedure, vortexing and centrifugation of sample following addition of 60% trichloroacetic acid. Cephalexin was used as an internal standard (I.S.). The HPLC analysis was carried out on a Capcell Pak C 18 analytical column with a mobile phase of 50 mM ammonium formate buffer/pH 3.5 and acetonitrile (90:10, v/v). No interference was observed near the peaks of cefroxadine and I.S. The calibration curve was linear over the range of 0.5–40 μg/mL and the lower limit of quantification (LLOQ) was 0.5 μg/mL. The method was validated with excellent sensitivity, accuracy, precision and stability. This assay was successfully applied to determine the pharmacokinetic parameters of cefroxadine in Korean healthy volunteers after an oral administration of two 250 mg cefroxadine capsules. As a result, the plasma half-life was 1.00 ± 0.26 h and the mean AUC 0–6 h was 46.25 ± 6.41 μg h/mL. The maximum plasma concentration ( C max) of 17.62 ± 4.87 μg/mL reached 1.44 ± 0.39 h after administration.

Analysis of Lipid Peroxidation and??Electron Microscopic Survey of??Maturation Stages during Human Cataractogenesis

Morphological and biophysical techniques described in this study have shown that membrane derangement occurs in human cataractous lenses. The data suggest that these disruptions were globules, vacuoles, multilamellar membranes and clusters of highly undulating membranes. Deleterious structural damage of the lens fibre cell plasma membranes serve as the primary light-scattering centres that cause the observed lens opacity. Nuclear cataract, a major cause of loss of lens transparency in the aging human, has been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. Phospholipid molecules modified by oxygen accumulate in the lipid bilayer, change its geometry and impair lipid-lipid and protein-lipid interactions in lenticular fibre membranes. Lipid peroxidation (LPO) is a causative and pathogenic factor in cataract. Increased concentrations of primary molecular LPO products (diene conjugates, lipid hydroperoxides, oxy-derivatives of phospholipid fatty acids) and end-fluorescent LPO products have been detected in the lipid moieties of aqueous humour samples and human lenses obtained from patients with senile and complicated cataracts as compared with normal donors. In the present study, a rapid and simple high-performance liquid chromatographic (HPLC) assay for determination of imidazole-containing dipeptides in the aqueous humour of the eye was developed. The method was applied to determine the pharmacokinetic parameters and the time-course of N-acetylcarnosine and L-carnosine-related product in the eye, following a single dosage of topical ocular administration of peptide. Utilising data from pharmacokinetic studies and the specific purity of the N-acetylcarnosine (NAC) ingredient as a source of the pharmacological principle L-carnosine, we have created an ophthalmic time-release prodrug form including the US FDA-approved carboxymethylcellulose lubricant and other essential ingredients (Can-C, private label Nu-Eyes). This formulation increases the intraocular absorption of L-carnosine in the aqueous humour and optimises its specific antioxidant activity in vivo while reducing the toxic effects of lipid peroxides on the crystalline lens. L-carnosine that enters the aqueous humour can accumulate in the lens tissue for a reasonable period of time. The presence of L-carnosine in transparent crystalline lenses during normal aging was detected and its concentration in this case was about 25 microM. At different stages of cataract development, the level of L-carnosine drastically decreased, reaching about 5 microM in ripe human cataracts. However, administration of pure L-carnosine (1% solution) to the rabbit eye (instillation or subconjunctival injection) does not lead to accumulation of this natural compound in the aqueous humour at the time level over 30 minutes at a concentration exceeding that in placebo-treated matched eyes, and its effective concentration is exhausted more rapidly. Use of NAC prodrug eye drops optimises the clinical effects of L-carnosine in the treatment of ophthalmic disorders (such as prevention and reversal of cataracts in human and animal [canine] eyes). The data provided predict a clinical effect with NAC ophthalmic prodrug, and show that the magnitude and duration of this effect are directly related to the bioavailability of L-carnosine released from NAC in the aqueous humour of the anterior eye segment. The ophthalmic NAC drug shows promise in the treatment of a range of ophthalmic disorders that have a component of oxidative stress in their pathogenesis (including cataract, glaucoma, dry eye, vitreous floaters, inflammatory disorders, and corneal, retinal and systemic diseases [such as diabetes mellitus and its ophthalmic complications]). There is a need for further and better collaboration between Innovative Vision Products' cataract control and ophthalmic services, improved education of people affected by cataract, a commitment that N-acetylcarnosine eye drops will be the preferred treatment before orthodox cataract surgery is attempted, and consideration of outcomes and a possible role of the NAC drug cataract treatment as source of referral for orthodox surgical, ophthalmic and optometric services.

A validated HPLC method with ultraviolet detection for the determination of buformin in plasma.

A sensitive and rapid high-performance liquid chromatographic assay is developed and validated for the determination of buformin in plasma. After addition of metformin as the internal standard, the analytes were deproteinated with acetonitrile, washed with dichloromethane, and the resulting supernatant injected. Chromatography was performed at ambient temperature by pumping a mobile phase of 0.03 m diammonium hydrogen phosphate buffer (pH 7, 250 mL) in methanol (750 mL) at a fl ow rate of 1 mL/min through a silica column. Buformin and metformin were detected at 236 nm, and eluted 9.8 and 15.4 min, respectively. No endogenous substances were found to interfere. Calibration curves were linear over the concentration range of 20-2000 ng/mL. The limit of quantitation was 20 ng/mL. The intra- and inter-day relative standard deviation (RSD) was 8.3%, or less, and the accuracy was within 10.1% of the relative error (RE). The method is suitable in pharmacokinetic investigation of buformin.

Determination of clofazimine in leprosy patients by high-performance liquid chromatography.

An original, simple, specific, and rapid high-performance liquid chromatography assay for the determination of clofazimine in human plasma is presented. The procedure consists of extracting the drug and the internal standard (medazepam) from 0.5 mL plasma with dichloromethane/diisopropyl ether (1:1, v/v) at pH 3.0, after precipitating the proteins with methanol. The drugs were then quantitated on a reversed-phase C8 using a mobile phase consisting of a mixture of methanol/0.25 N sodium acetate buffer at pH 3.0 (74:26, v/v). The flow-rate and wavelength were set at 1 mL/min and 286 nm, respectively. The precision, linearity, and limit of quantitation of the method were within acceptable limits. The method was considered adequate and could be applied in studies involving blood level monitoring and pharmacokinetics in leprosy patients.

Rapid high-performance liquid chromatographic assay of dorzolamide in rabbit aqueous humor.

A rapid high-performance liquid chromatographic method using a C(18) reversed-phase column (Hypersil ODS) with UV detection at 254 nm and a simple pre-treatment of samples is presented for the analysis of dorzolamide, a carbonic anhydrase inhibitor, in rabbit aqueous humor. A water solution containing 2% ZnSO(4) small middle dot7H(2)O was used to deproteinize aqueous humor samples. The mobile phase consisted of 7% CH(3)CN and 93% of a solution containing 1% TEA adjusted to pH = 3.5 with H(3)PO(4). Paracetamol was found to be a suitable internal standard. The standard curves were linear in the detection range. The precision and the accuracy were <5% for both intra- and inter-day assays.

Rapid screening of urinary proline–hydroxyproline dipeptide in bone turnover studies

In a recent report [J. Chromatogr. B 678 (1996) 165] a urinary hydroxyproline-containing peptide has been preliminarily suggested as a possible alternative to hydroxyproline (HP) determination in bone resorption studies. For this purpose a simple and practical procedure was developed for a rapid high-performance liquid chromatographic (HPLC) assay of the peptide in non-hydrolyzed urine samples. Hundreds of randomly selected urine samples were assayed for both the peptide and HP, the latter in hydrolyzed urine, and a high correlation between them was found. The promising results prompted us to search for the postulated biomarker of bone resorption in urine samples of postmenopausal women examined as osteoporosis suspects. As an alternative to the HPLC determination, an equally rapid procedure has been developed for the peptide assay using capillary gas chromatography (GC) with flame ionization detection (FID). By means of a solid-phase and a liquid–liquid phase extraction, involving ethyl chloroformate (ECF) as the derivatizing agent, two dipeptides and some urinary amino acids could be analyzed within 5 min. A high correlation between both HPLC and GC peptide assay was confirmed ( r=0.944) and the compound was identified as proline–hydroxyproline (PHP) dipeptide.

Rapid determination of valaciclovir and acyclovir in human biological fluids by high-performance liquid chromatography using isocratic elution

A rapid high-performance liquid chromatographic assay with isocratic elution is developed for the simultaneous quantification of valaciclovir (VACV) prodrug and its active converted compound, acyclovir (ACV), in biological fluids of treated patients. For serum, the samples are deproteinized with perchloric acid in presence of 1-methylguanosine as the internal standard (IS). For urine and dialysis liquid, the samples are diluted with a mobile phase containing the IS, then filtered. VACV, ACV and the IS are separated on a SymmetryShield™ RP-8 column with acetonitrile–ammonium phosphate buffer as the mobile phase and detected at 254 nm. The chromatographic time is about 12 min. The relative standard deviations (RSD) of VACV and ACV standards are between 0.5 and 3.5%. Most endogenous nucleosides and their metabolites, psychotropic drugs and drugs of abuse are shown not to interfere with this technique. The method has been applied to study the pharmacokinetics of VACV and ACV in serum, dialysis liquid and urine of renal failure patients on continuous ambulatory peritoneal dialysis (CAPD) under oral treatment of VACV.

A simplified and rapid high-performance liquid chromatographic assay for ketoprofen in isopropyl myristate.

A high-performance liquid chromatographic (HPLC) procedure for quantitating ketoprofen in isopropyl myristate (IPM), a compound widely used as a receptor medium in drug diffusion studies of topical aqueous-based formulations, is developed. Previously reported HPLC assays for ketoprofen in IPM have employed relatively complex and tedious methods for purifying the IPM prior to injection onto the HPLC column. The present assay method utilizes a direct injection of the IPM-based sample onto a new reversed-phase ODS column and employs ultraviolet detection at 265 nm. Propyl paraben is employed as the internal standard. The mobile phase consists of acetonitrile-methanol-water (36:54:10, v/v/v) at a flow rate of 1.2 mL/min. The calibration curves are linear (correlation coefficient r > or = 0.988) over concentration ranges of 0.625-10 micrograms/mL and 6.25-100 micrograms/mL. The within-day and between-day precision exhibit coefficients of variation of 1.3-3.3%, and the accuracy (reported as relative error of the mean) varies from -1.9% to 0.6%. The retention times for ketoprofen and propyl paraben are approximately 2.3 and 3.3 min, respectively. The total run time per sample is approximately 7 min. The minimum quantitatable concentration is approximately 0.625 microgram/mL. The assay is stability-indicating, rapid, reproducible, sensitive, and readily adaptable for assaying other non-steroidal anti-inflammatory drugs.

A rapid high-performance liquid chromatographic assay for cefepime, cefpirome and meropenem

A rapid high-performance liquid chromatographic assay for cefepime, cefpirome and meropenem

Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes.

A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C8 column with a mobile-phase of acetonitrile-0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25-400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.

A rapid, specific, and sensitive method for the determination of acetylation phenotype using dapsone.

An original, simple, specific, and rapid high-performance liquid chromatography assay for the determination of dapsone and monoacetyldapsone in human plasma is presented. The procedure consists of extracting the drugs and the internal standard (phenacetin) from 1 mL plasma with ethyl ether at alkaline pH. A liquid chromatograph equipped with a reversed-phase 5-microm C8 analytical column was used. The drugs were eluted with a mixture of water and methanol (70:30, v/v). Flow rate and wavelength were set at 1 mL/min and 286 nm, respectively. The precision, linearity, and limit of quantitation of the method were within acceptable limits. The method was considered adequate and has been used for the determination of the acetylator phenotype in population studies.

Measurement of plasma ketoprofen by a rapid high-performance liquid chromatography assay.

The authors developed a high performance liquid chromatography assay for the determination of plasma ketoprofen concentration using a single-step extraction, a short chromatographic separation of 3 min, and a sample volume of 50 microliters. The assay possessed linearity up to 500 mg/l, sensitivity down to at least 1 mg/l, average recovery of 100.1%, and run-to-run precision (n = 20) of 4.6% at a level of 10 mg/l and 2.2% at a level of 40 mg/l. Furthermore, the assay was free of interference from 51 prescription and over-the-counter medications. The authors conclude that the method described here is ideally suited for the determination of ketoprofen concentration in a clinical laboratory setting for the purpose of therapeutic monitoring and assessment of toxicity.

Rapid high-performance liquid chromatographic assay for salicylic acid in plasma without solvent extraction.

The in vivo measurement of highly reactive free radicals, such as the hydroxyl radical (oOH), in humans is very difficult, if not impossible. Specific markers, based on the ability of oOH to attack aromatic molecules and produce hydroxylated compounds, are under investigation. In vivo radical metabolism of salicylic acid produces two main hydroxylated derivatives: 2,3- and 2,5-dihydroxybenzoic acid (DHBA). The measurement of 2,3-DHBA, following oral administration of salicylic acid or its acetylated form (aspirin), is proposed for the assessment of in vivo oxidative stress. The intensity of oxidative stress is a function of the ratio of dihydroxylated derivatives to salicylic acid rather than the absolute dihydroxylated derivatives levels. Consequently, a simple, accurate, and sensitive assay of the salicylic acid level in plasma is needed to investigate the in vivo free radical production. In this work, a rapid and sensitive method is presented that is useful for the quantitation of salicylic acid in biological fluids. The methodology uses high-performance liquid chromatography with spectrophotometric detection for the identification and quantitation of salicylic acid without organic extraction. A detection limit of less than 5 mumol is achieved with spectrophotometric detector responses that are linear over at least 6 orders of magnitude. Plasma concentrations of salicylic acid determined by the present technique are reported following the administration of 1000 mg aspirin in 20 healthy subjects.

Determination of 3,4-Dihydroxyphenylacetic Acid and 5-Hydroxyindoleacetic Acid in Human Plasma by a Simple and Rapid High-performance Liquid Chromatography Assay

AbstractIn order to study the pharmacodynamic effects of drugs on dopamine and serotonin metabolism, a reversed-phase HPLC assay coupled with electrochemical detection (ECD) for measuring plasma concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hy-droxyindoleacetic acid (5-HIAA) was developed. The system was operated isocratically using a mobile phase of aqueous 0.03MKH2PO4 buffer containing 0.15mM EDTA in methanol (8.75:1.25), with a final pH of 4.0. The flow rate was set at 1.5mL/min, and potentials at +450mV. Using a signal to noise ratio of >3, the minimum detection limit assessed by direct on-column injection of a standard solution for DOPAC and 5-HIAA was <1pg. The assays were linear from basal concentrations (1–10ng/mL) to 100ng/mL. The intra- and interassay variations were <10% and <20%, respectively.

Rapid, specific and sensitive method for isoniazid determination in serum

An original simple, specific and rapid high-performance liquid chromatographic assay for the determination of isoniazid (INH) in human serum is presented. The drug was extracted from the serum by protein precipitation with 30% (w/v) trichloroacetic acid, then the drug was reacted with the coupling reagent, trans-cinnamaldehyde, to form a derivative absorbing at 340 nm. A 20-μl aliquot was injected into the chromatograph after neutralization with 1 M KOH solution. A liquid chromatograph equipped with a reversed-phase 30-μm C 18 precolumn linked to a 4-μm C 18 analytical column was used. The drug was eluted with a mixture of acetonitrile-water-triethylamine-acetic acid (400:600:2:1, v/v), pH value was 5±1. Flow-rate and wavelength were set at 1 ml/min and 340 nm, respectively. The extraction recoveries from human serum averaged 100% for INH at concentrations of 1, 2 and 4 mg/1. The coefficients of variation for three different concentrations of INH in serum in the within-day study varied between 1.2 and 3.5%, whereas those in the day-to-day study varied between 2.8 and 4.3%.