High-performance liquid chromatography determination of praziquantel enantiomers in human serum using a reversed-phase cellulose-based chiral stationary phase and disc solid-phase extraction

A sensitive HPLC method for the quantification of praziquantel enantiomers in human serum is described. The method involves the use of a novel disc solid-phase extraction for sample clean-up prior to HPLC analysis and is also free of interference from trans-4-hydroxypraziquantel, the major metabolite of praziquantel. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OJ-R) under isocratic conditions using a mobile phase consisting of 0.1 M sodium perchlorate–acetonitrile (66:34, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R-(−)- and S-(+)-praziquantel enantiomers were in the range of 84–89% at 50–500 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 3–8% and 1–8% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.2–5% and 0.3–8% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 10–600 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 5 ng/ml (S/N=2).     

Plasma concentrations of temazepam,a 3-hydroxy benzodiazepine,determined by electron-capture gas—liquid chromatography

A simple and sensitive high-performance liquid chromatographic assay of methotrexate (MTX) and its two active metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-di-amino-N¹⁰-methylpteroic acid (APA) in plasma, saliva and urine was developed. The method involved deproteinization with acetonitrile followed by addition of isoamyl alcohol and ethyl acetate. After extraction the sample was chromatographed on a cation-exchange column and monitored at 313 nm. The retention times were 5, 7 and 9 min and detection limits 20, 10 and 5 ng/ml for 7-OH-MTX, MTX and APA, respectively. For concentrations greater than 100 ng/ml one-step deproteinization of 0.1 ml sample with 0.25 ml acetonitrile was satisfactory for sample preparation. The method has been evaluated in samples from patients and rabbits receiving MTX.     

Development of sensitive high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent for determination of bisphenol A in plasma samples

A sensitive HPLC method for determination of bisphenol A (BPA) in plasma samples using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a fluorescence labeling reagent was developed. The fluorescence labeling reaction was completed within 10 min at room temperature. DIB-Cl reacts with the phenolic hydroxyl group of BPA in the presence of triethylamine (TEA). The DIB-Cl derivative of BPA (DIB-BPA) was separated within 30 min with an ODS column using acetonitrile–water (90:10, v/v) as the isocratic eluent. Calibration graphs were linear over the range of 1.0–100 ng/ml (r=0.999). The detection limit of DIB-BPA was 0.05 ng/ml (2.5 pg) at a signal-to-noise ratio of 3. The relative standard deviations (RSDs) of the method for between-run were 1.0–5.0%. The analytical recoveries of known amounts (1.0 and 100 ng/ml) of BPA-spiked rabbit plasma were around 95%.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching : I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17x4.6 mm I.D.), filled with C₁₈ Corasil 37–53 μm. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-1000 ng/ml was between 1.0 and 4.7% for all compounds.     

Determination of paclitaxel and metabolites in mouse plasma,tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1–0.2 g/ml) in bovine serum albumin (40 g/I) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200–1000 μl of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut column. 2′-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 ot 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 μl of sample. In tissues, faeces and urine the LLQs are 25–100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 μl (faeces: 200 μl) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25–100 000 ng/ml for paclitaxel and 25–500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.     

Natural Occurrence of Ochratoxin A Contamination in Commercial Black and White Pepper Products

The concentration of ochratoxin A (OTA) in 120 commercial pepper (84 pre-packed and 36 bulk samples), which consist of local and imported white and black pepper in powder and seed form in Malaysia were determined. The objective of the study was to investigate and compare OTA concentration in black pepper and white pepper being commercialized in Malaysia. Determination method was based on HPLC with fluorescence detection coupled with immunoaffinity column clean-up step. Mobile phase consisted of acetonitrile–water–acetic acid (49.5:49.5:1.0, v/v/v), and flow rate was 1 ml/min. The LOD was 0.02 ng/g, and the average recovery values of OTA ranged from 79.5 to 92.0% in black pepper and 81.2–90.3% in white pepper. A total of 57 samples (47.5%) were contaminated with OTA ranging from 0.15 to 13.58 ng/g. The results showed that there was a significant difference between type of pepper and brands. OTA concentration in black pepper was significantly higher than white pepper (p < 0.05). The highest concentration of ochratoxin, 13.58 ng/g, was detected in a sample of black pepper seed followed by 12.64 ng/g in a sample of black pepper powder, both were bulk samples purchased from open market.     

High-performance liquid chromatographic method for the simultaneous determination of HIV-1 protease inhibitors indinavir,saquinavir and ritonavir in plasma of patients during highly active antiretroviral therapy

A new high-performance liquid chromatographic method for the simultaneous determination of indinavir, saquinavir and ritonavir in human plasma is described. Quantitative recovery following liquid–liquid extraction with diethyl ether from 500 μl of human plasma was achieved. Subsequently, the assay was performed with a linear gradient starting at 67 mM potassium dihydrogenphosphate–acetonitrile (65:35 to 40:60, v/v) as a mobile phase, a Phenomenex C₁₈ column and UV detection at 240 and 258 nm, respectively. Linear standard curves were obtained for concentrations ranging from 75 to 20 000 ng/ml for indinavir, from 10 to 6000 ng/ml for saquinavir, and from 45 to 30 000 ng/ml for ritonavir. The calculated intra- and inter-day coefficients of variation were below 6%.     

High-performance liquid chromatographic analysis of mibefradil in dog plasma and urine

The objective of the study was to develop a sensitive and specific assay for studying the pharmacokinetics of a novel calcium antagonist, a benzimidazolyl-substituted tetraline derivative, mibefradil (I) in the dog. The assay involves liquid-liquid extraction of a biological sample, reversed-phase HPLC separation and fluorescence detection (λ_ex = 270 nm and λ_em = 300 nm) of a sample components. Each sample was eluted with a mobile phase pumping at a flow-rate of 2 ml/min. The mobile phase composition was a mixture of acetonitrile and aqueous solution (38:62, v/v). The aqueous solution contains 0.0393 M KH₂PO₄ and 0.0082 M Na-pentanesulphonic acid. The retention times were 10.7 min for I, and 12.2 min for internal standard Ro 40–6792. Calibration curves with concentrations of I ranging from 10 to 500 ng/ml were linear (r² > 0.99). The detection limit for I was 0.5 ng/ml when 0.5 ml of plasma or urine was used. Intra- and inter-day accuracy and precision were within 10%. The assay was successfully applied to the pharmacokinetic studies of I in dogs.     

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Simultaneous determination of selegiline-N-oxide,a new indicator for selegiline administration,and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)–electrospray ionization mass spectrometry (HPLC–ESI–MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C₁₈ cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.×150 mm) for HPLC separation and ESI–MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile–10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10–20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users’ urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.     

Routine and sensitive method for determination of nicorandil in human plasma developed for liquid chromatography with ultraviolet and m0ass spectrometric detection

A rapid and sensitive method using HPLC has been developed for the quantification of nicorandil (SG-75) in human plasma samples for routine bioequivalence studies. The sample preparation needs two liquid–liquid extractions, first with CH₃Cl and HClO₄ as denaturation reagent and second with addition of ethyl acetate and Na₂CO_3(aq). Detection wavelength was 256 nm. The obtained correlation coefficient for weighted linear curve in the range from 5.0 to 300 ng/ml was higher than 0.9950. The limit of quantitation (LOQ) was established at 5.0 ng/ml. The HPLC separation was accomplished on Nucleosil Phenyl (5 μm) stainless steel column within 7 min. The mixture of 0.01 M ammonium acetate buffer (pH 6.2) and acetonitrile 10:3 (v/v) was used as the mobile phase. The same separation method was examined on HPLC–MS system. Using this system, the LOQ was established at 1.0 ng/ml and the linearity was obtained in the range from 1.0 to 150 ng/ml.     

High-performance liquid chromatographic–tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

A sensitive and specific high-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS–MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC–MS–MS. Linearity was given in the tested range of 50–5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50–5000 ng/ml was 1.47–6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37°C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.     

Analysis of cisapride in neonatal plasma using high-performance liquid chromatography with a base-stable column and fluorescence detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the prokinetic drug cisapride is described. Alkalinised samples of plasma (100 μl) were extracted with 1.0 ml of 10% (v/v) isopropanol in chloroform, dried, redissolved in mobile phase and injected. Chromatography was performed at 20°C by pumping a mobile phase of acetonitrile (370 ml) in pH 5.2, 0.02 M phosphate buffer (630 ml) at 1.0 ml/min through a C₈ Symmetry column. Cisapride and the internal standard were detected by fluorescence monitoring at 295 nm (excitation) and 350 nm (emission), and were eluted 5 min and 8 min, respectively, after injection. Calibration plots in bovine serum albumin (3% w/v) were linear (r > 0.999) from 5 to 250 ng/ml. Intra-day and inter-day precision (C.V.) was 9.5%, or less, and the accuracy was within 5.5% of the nominal concentration over the range 8–200 ng/ml. Total assay recovery was above 82%. Endogenous plasma components, major cisapride metabolite (norcisapride), and other durgs used in neonatal pharmacotherapeutics did not interfere.     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Novel liquid chromatographic assay for the low-level determination of apomorphine in plasma

A novel HPLC assay which is rapid, reproducible and sensitive has been developed for the analysis of apomorphine in plasma. The assay incorporates boldine as an internal standard, and uses solid-phase extraction on C₁₈ mini-columns for sample clean-up and concentration, so enabling quantitation of apomorphine at 500 pg/ml using fluorescence detection (λ_ex 270 nm, λ_em). The HPLC assay comprised a 25 cm-long Techopakk C₁₈ column and a mobile phase of (0.25 M sodium dihydrogen phosphate plus 0.25% heptane sulphonic acid, to pH 3.3 with orthophosphoric acid) containing 30% (v/v) methanol and 0.003% (w/v) EDTA, run at a flow-rate of 1.5 ml/min. Calibration plots prepared in plasma were linear over the range 1–30 ng/ml, (limit of quantitation (LOQ)=490 PG/ML) with R.S.D. of 0.05% and R.E. of 5.0% at the level of 1 ng/ml. Preliminary pharmacokinetic data from two patients given apomorphine by 12 h subcutaneous infusion (patient A dose=35 mg and patient B dose=141 mg) showed apomorphine elimination from plasma to fit a two-compartment model, with initial half-lives of 8.2 and 46.6 min, elimination half-lives of 76.4 and 166.5 min and area under the plasma concentration-time curve (AUC) values of 236 and 405 ng h/ml, respectively.     

Determination of piromidic acid residues in trout muscle tissue and in urine by liquid chromatography with post-column modification of pH and fluorimetric detection

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C₁₈ column was used with acetonitrile–4·10⁻⁴ M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm (λ_ex 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.     

Pharmacokinetic applicability of a validated liquid chromatography tandem mass spectroscopy method for orally administered curcumin loaded solid lipid nanoparticles to rats

A simple and sensitive validated LC–MS/MS analytical method was used for determination of curcumin in rat plasma, using nimesulide as internal standard. Analyses were performed on an Agilent LC–MS/MS system using a Chromolith rod? and isocratic elution with acetonitrile:10 mM ammonium acetate buffer (pH 3.5) (80:20, v/v) at a flow rate of 0.8 ml/min with a total run time of 3 min and an overall recovery of 77.15%. A triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the negative mode was used. Calibration curve in plasma spiked with varying concentration of curcumin were linear over the concentration range of 10–2000 ng/ml with determination coefficient >0.99. The lower limit of quantification was 10 ng/ml. Intra and inter-day variability's (RSD) for extraction of curcumin from plasma were less than 10% and 15% respectively and accuracy was 102.43–108.5%. Multiple reaction monitoring was used to monitor the transition for curcumin (m/z; 367/217 [M?H]^?) and IS (m/z; 307/229). The method was applied for determining curcumin concentration in plasma after peroral administration of 50 mg/kg of free curcumin (C-S) or curcumin loaded solid lipid nanoparticles (C-SLNs) to rats. Results established selectivity and suitability of the method for pharmacokinetic studies of curcumin from C-SLNs.     

Extraction and determination of oxybutynin in human bladder samples by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method is described for the determination of oxybutynin (OXB) in human bladder samples. Following homogenization, tissue samples underwent double extraction with hexane and eventually were concentrated by freeze–drying before analysis. Chromatographic separation was performed with a mobile phase of acetonitrile–water–1 M ammonium acetate, pH 7.0 (85:13:2, v/v/v) at a flow-rate of 0.5 ml/min and double (electrochemical and UV) detection was applied. The retention time of oxybutynin eluting peak was around 18 min. Using a standard curve range of 10 to 500 ng/ml the quantification limit with electrochemical detection was 5 ng/ml with an injection volume of 100 μl. Within-day and day-to-day relative standard deviation values were 4.9 and 9.81%, respectively, while a 94% accuracy and a 72% recovery was attained. We applied this method to compare the OXB levels into bladder wall tissue samples after passive diffusion and after electromotive drug administration (EMDA), using a two-chambered poly(vinyl chloride) diffusion cell designed and developed in our laboratory. The results obtained show that EMDA enhanced OXB penetration into bladder wall and that this novel way of local drug administration can be potentially used in patients with neurogenic bladder dysfunction or urinary incontinence.