Determination of spectinomycin hydrochloride and its related substances by HPLC-ELSD and HPLC-MSn

A new and simple high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method for the determination of spectinomycin hydrochloride and its related substances was developed. The column was Agilent SB-C(18) (250 mm x 4.6 mm, 5 microm).The mobile phase was 25 mM trifluoroacetic acid. The drift tube temperature was 40 degrees C. The pressure of nebulizing gas was 3.5 bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07-3.8 mg/ml (r = 0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6 microg/ml. The method is simple and rapid, and the results are accurate and reproducible. The HPLC-MS(n) method was used to characterize the structures of impurities contained in the spectinomycin. In positive mode, impurities were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. The possible structures of impurities C and D in spectinomycin were deduced based on the HPLC-MS(n) data.     

Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method was developed to determine free didanosine concentrations in human serum. An ultrafiltration technique was used to recover didanosine from the samples. Didanosine was analyzed using a 150 mm × 3.9 mm I.D. Nova-Pak phenyl column and a mobile phase of 0.02 M sodium citrate (pH 5)-isopropanol (97.5:2.5, v/v) with detection set at 250 nm. Linearity was verified from 25 to 3000 ng/ml. The limit of detection at a signal-to-noise ratio of 3 was 25 ng/ml. The mean recovery of didanosine added to serum at 50, 100, 250 and 750 ng/ml was 97.4%, 97.3%, 92.9% and 95.4%, respectively. A within-day variation of 3.6% at 50 ng/ml and 1.7% at 250 ng/ml, and a day-to-day variation of 9.3% at 50 ng/ml and 3.6% at 230 ng/ml were found. Stability studies indicated that didanosine is stable in serum for at least 8.5 months at 20°C, 4°C and −20°C.     

High-performance liquid chromatographic determination of methoxsalen in plasma after liquid—solid extraction

An improved method suitable for the determination of 8-methoxypsoralen in the range 50–1500 ng/ml in the plasma of psoriatic patients undergoing PUVA (psoralens and long-wave ultraviolet light) therapy is proposed. A 5-ml aliquot of plasma containing sodium citrate as anticoagulant was centrifuged, griseofulvin was added as internal standard and the sample was denatured with acetonitrile. The supernatant was applied to C₁₈ cartridges and 8-methoxypsoralen was eluted with methanol. The evaporated eluate was reconstituted in the mobile phase for high-performance liquid chromatography (HPLC) and applied to the HPLC column: mobile phase, acetonitrile—0.01 M phosphoric acid (34:66); flow-rate, 1 ml/min; temperature, 40°C; column, Spherisorb 5 ODS, 100 mm × 4.6 mm I.D., 5 μm particle size; UV detection at 248 nm; detection limit, 15 ng/ml of plasma.     

Ion-pair high-performance liquid chromatographic assay method for the assessment of clarithromycin stability in aqueous solution and in gastric juice

A simple and selective ion-pair HPLC method has been developed for the analysis of clarithromycin in aqueous solutions and in gastric juice. A Hypersil ODS 5-μm (150 × 4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer (pH 4.6) containing 5 mM 1-octanesulphonic acid (50:50, v/v). The column temperature was 50°C and detection was by UV absorption (210 nm). The limits of detection of 50-μl samples were 0.4 μg/ml (aqueous) and 0.78 μg/ml (0.5 ml gastric juice) or better. The assay was linear in the range of 1.56 to 100 μg/ml with r² values greater than 0.99. The recovery from the gastric juice samples was 98.5±2.9%. The method was applied successfully to determine the stability of clarithromycin in 0.01 M HCl and gastric juice.     

Stereospecific determination of mefloquine in biological fluids by high-performance liquid chromatography

A sensitive stereoselective HPLC method was developed for determination of mefloquine (MFQ) enantiomers in plasma, urine and whole blood. The assay involved liquid-liquid extraction of MFQ from biological fluids with a mixture of hexane and isopropanol in the presence of sodium hydroxide and derivatization of the residue by (+)-(S)-naphthylethylisocyanate (NEIC) as chiral derivatizing reagent. Separation of the resulting diastereomers was performed on a silica normal-phase column using chloroform-hexane-methanol (25:74:1) as the mobile phase with a flow-rate of 1 ml/min. Using 200 μl of plasma or whole blood, the limit of determination was 0.2 μg/ml with UV detection for both enantiomers. The limit of determination in 500 μl of urine was 0.08 μg/ml with UV detection.     

A validated enantiospecific method for determination and purity assay of clopridogrel

A new and accurate HPLC method using β‐cyclodextrin chemically bonded to spherical silica particles as chiral stationary phase (CSP) was developed and validated for determination of S‐clopidogrel and its impurities R‐enantiomer and S‐acid as a hydrolytic product. The effects of acetonitrile and methanol content in the mobile phase and temperature on the resolution and retention of enantiomers were investigated. A satisfactory resolution of S‐clopidogrel active form and its impurities was achieved on ChiraDex® column (5 μm, 4 × 250 mm) at a flow rate of 1.0 ml/min and 17°C using acetonitrile, methanol and 0.01 M potassium dihydrogen phosphate solution (15:5:80 v/v/v) as mobile phase. The detection wavelength was set at 220 nm. The method was validated in terms of accuracy, precision, linearity, and robustness. The limit of detection for R‐enantiomer and S‐acid were 0.75 and 0.09 μg/ml, respectively, injection volume being 20 μl. Finally, the molecular modeling of the inclusion complexes between the analytes and β‐cyclodextrin was performed to investigate the mechanism of the enantiorecognition and to study the quantitative structure–retention relationships. Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Assay for etoposide in human serum using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

An HPLC assay for etoposide in human serum was developed. Serum, spiked with podophyllotoxin (internal standard), was treated with sodium dodecyl sulphate prior to solid phase extraction. Analysis was performed on a 300×3.9 mm Bondclone 10 C18 column coupled with a fluorometric detector (λ_ex 230 nm, λ_em 330 nm). The retention times for etoposide and podophyllotoxin were 14 and 28 min respectively. The range of assay was 0.5 to 20 μg/ml with a detection limit of 0.2 μg/ml. This assay is suitable for use in clinical studies with etoposide.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithio-carbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 μl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150×3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 μg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 μg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 μg/ml in plasma (500 μl) using a weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ration values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 μg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

Simple,rapid and sensitive method for the determination of indomethacin in plasma by high-performance liquid chromatography with ultraviolet detection

A micro method for determination of indomethacin in plasma was developed. Following deproteinization of plasma with acetonitrile containing internal standard (mefenamic acid), the separation of indomethacin and internal standard was achieved by high-performance liquid chromatography using a 7 μm LiChrosorb-RP18 column (250×4 mm I.D.) at 50°C. The mobile phase was 6 mM phosphoric acid–acetonitrile (50:50). The flow-rate was kept at 2.0 ml/min and the column effluent was monitored at 205 nm. The coefficients of variation of the method estimated at 0.2 and 1.0 μg/ml were 4.2 and 2.3%, and the detection limit of the drug was about 0.05 μg/ml (S/N=5). The method requires minimum pretreatment of the plasma with a small sample volume (25 μl), and is very suitable for therapeutic drug monitoring of indomethacin in premature infants with symptomatic patent ductus arteriosus.     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.     

Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) assay for biperiden in bulk form and pharmaceutical dosage forms

Current compendial (USP) methods of assay for the analysis of biperiden in bulk form and pharmaceutical dosage forms involve the use of titrimetric and spectrophotometric procedures, respectively. These are non-selective and non-stability-indicating techniques. In this work, a stability-indicating high performance liquid chromatographic assay procedure has been developed and validated for biperiden. The liquid chromatographic separation was achieved isocratically on a symmetry C8 column (150 mm x 3.9 mm i.d., 5 microm particle size) using a mobile phase containing methanol-buffer (50:50, v/v, pH 2.50) at a flow rate of 1 ml/min and UV detection at 205 nm. The buffer was composed of sodium dihydrogen phosphate (50 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The method was linear over the concentration range of 0.5-25 microg/ml (r=0.9998) with a limit of detection and quantitation 0.03 and 0.1 microg/ml, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay biperiden in bulk form and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of biperiden and the assay is thus stability-indicating.     

Simultaneous determination of diastereoisomeric and enantiomeric impurities in (1R, 3R)-1-(1,3-benzodioxol-5-yl)-2-(chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester a key intermediate of tadalafil by chiral high-performance liquid chromatography

(1R, 3R)-1-(1, 3-Benzodioxol-5-yl)-2-(chloroacetyl)-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester ((1R, 3R)-Cpe) is a key intermediate used in the synthesis of tadalafil, a highly selective phosphodiesterase type-5 inhibitor. In the present study, a chiral high-performance liquid chromatography method was developed for the simultaneous determination of diastereoisomeric and enantiomeric impurities in (1R, 3R)-Cpe. Separation was performed on an Ultron ES-OVM chiral column (150 mm × 4.6 mm, 5 μm,) with a guard column at a column temperature of 30°C. The gradient elution used was acetonitrile (solvent A) and water (solvent B), and the following elution program was used at a flow rate of 1 ml/min: 0-5 min (80% B), 5-10 min (80-60% B), 10-12 min (60% B). The detection wavelength was 220 nm. The four isomers of Cpe were baseline separated in 12 min. The results of method validation indicated that the method was specific and sensitive and was suitable for the quality control of diastereoisomeric and enantiomeric impurities in (1R, 3R)-Cpe.     

Ion chromatographic determination of taurine in medicine, nutrient capsule and human urine with electrochemical detection

A very simple and sensitive method for the determination of taurine by ion chromatography with electrochemical integrated pulsed amperometry is firstly described. Taurine was determined using 160 mmol/l NaOH as eluent and a Dionex CarboPac™ PA1 separation column (250×4 mm I.D.) without the interference with ten kinds of common amino acids. The peak area response of taurine was linear in the range 0.1–20 μg/ml, the detection limit was 0.034 μg/ml. The method has been applied successfully in the determination of taurine in medicinal granule, nutrient capsule and human urine. The content determined in medicinal granule is consistent with that marked by the manufacturer.     

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development,validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.     

Sensitive assay for midazolam and its metabolite 1′-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1′-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 μm capillary C₁₈ column (150 mm×0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer–acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer–acetonitrile, 40:60. The flow-rate of the mobile phase was 16 μl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1′-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1′-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 μg/kg).     

A stability-indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets

A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC method was developed and validated for the simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate separation for AM, AT from their associated main impurities and their degradation products. Separation was achieved on a Perfectsil Target ODS-3, 5 microm, 250 mm x 4.6 mm i.d. column using a mobile phase consisting of acetonitrile-0.025 M NaH(2)PO(4) buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method was investigated in the range of 2-30 microg/ml (r=0.9994) for AT and 1-20 microg/ml (r=0.9993) for AM. The limits of detection were 0.65 microg/ml and 0.35 microg/ml for AT and AM, respectively. The limits of quantitation were 2 microg/ml and 1 microg/ml for AT and AM, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AT and AM and the assay can thus be considered stability-indicating.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 × 2.1 mm I.D., 5 μm Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate—acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 μl sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean ± S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 ± 1.2% and 91.4 ± 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Automated analysis of quetiapine and other antipsychotic drugs in human blood by high performance-liquid chromatography with column-switching and spectrophotometric detection

An automated HPLC method with column switching is described for the determination of quetiapine, clozapine, perazine, olanzapine and metabolites in blood serum. After clean-up on silica C8 material (20 microm particle size) drugs were separated on ODS Hypersil C18 material (5 microm; column size 250 mm x 4.6 mm i.d.) within 25 min and quantified by ultraviolet (UV) detection at 254 nm. The limit of quantification ranged between 10 and 50 ng/ml. At therapeutic concentrations of the drugs, the inter-assay reproducibility was below 10%. Analyses of drug concentrations in serum of 75-295 patients treated with therapeutic doses of the antipsychotic drugs revealed mean+/-S.D. steady state concentrations of 139+/-136 ng/ml for quetiapine, 328+/-195 ng/ml for clozapine, 48+/-27 ng/ml for olanzapine and 71+/-52 ng/ml for perazine. The method was thus suitable for routine therapeutic drug monitoring and may be extended to other drugs.     

Determination of non-protein bound phenylbutazone in bovine plasma using ultrafiltration and liquid chromatography with ultraviolet detection

A liquid chromatographic procedure using UV detection was coupled with ultrafiltration for the quantitation of free phenylbutazone in bovine plasma, in the range of 20 ng/ml to 2.0 μg/ml. Whole plasma samples (0.5 to 1 ml) were placed in a 2-ml centrifugal concentrator with a molecular-mass cut-off membrane of 10 000 and centrifuged at 4500 g for 2 h at 4°C using a fixed angle rotor. The ultrafiltrate was transferred to an LC vial with a 200-μl insert and 100 μl was injected into an LC system. The chromatographic system used a C₁₈ reversed-phase column connected to a UV detector set at 264 nm. The mobile phase was 0.2 M sodium phosphate buffer (pH 7)–methanol (1:1). Recoveries of phenylbutazone from protein-free plasma water fortified at levels of 20 ng/ml to 2 μg/ml ranged from 91 to 93%, with relative standard deviations (R.S.D.s) ranging from 1 to 4%. The concentration of incurred non-protein bound phenylbutazone obtained from a cow intravenously dosed twice with 2 g phenylbutazone, 8 h apart, was 111, 26 and 11 ng/ml for 2, 72 and 104 h post first phenylbutazone dose, respectively.