Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4′-hydrazino-2-stilbazole

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4′-hydrazino-2-stilbazole (4′H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis–trans isomerism at the proline–pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4′H2S is performed in a water–acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2–100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4′H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.     

Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatography–mass spectrometry-selected ion monitoring

A sensitive, selective, and reproducible GC–MS–SIM method was developed for determination of artemether (ARM) and dihydroartemisinin (DHA) in plasma using artemisinin (ART) as internal standard. Solid phase extraction was performed using C₁₈ Bond Elut cartridges. The analysis was carried out using a HP-5MS 5% phenylmethylsiloxane capillary column. The recoveries of ARM, DHA and ART were 94.9±1.6%, 92.2±4.1% and 81.3±1.2%, respectively. The limit of quantification in plasma was 5 ng/ml (C.V.≤17.4% for ARM and 15.2% for DHA). Calibration curves were linear with R²≥0.988. Within day coefficients of variation were 3–10.4% for ARM and 7.7–14.5% for DHA. Between day coefficients of variations were 6.5–15.4% and 7.6–14.1% for ARM and DHA. The method is currently being used for pharmacokinetic studies. Preliminary data on pharmacokinetics showed C_max of 245.2 and 35.6 ng/ml reached at 2 and 3 h and AUC_0–8h of 2463.6 and 111.8 ngh/ml for ARM and DHA, respectively.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.     

Determination of idarubicin and idarubicinol in rat plasma using reversed-phase high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of idarubicin and idarubicinol in rat plasma. Blood samples were analyzed from 16 rats which had received an intravascular dose of 2.25 mg kg⁻¹ idarubicin. After deproteinization with acetonitrile, the separation was performed with a LiChrospher 100 RP-18 column (5 μm), using fluorescence detection (excitation: 485 nm/emission: 542 nm). The mean recovery was 95.6% for idarubicin and 90.7% for idarubicinol, respectively. The detection limit was 0.25 ng ml⁻¹ using an injection volume of 50 μl. Daily relative standard deviation (RSD) was 3.2% (10 ng idarubicin/ml, n=10) and 4.4% (10 ng idarubicinol/ml, n=10).     

Quantification of mycophenolate mofetil in human skin extracts using high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC–MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C₁₈ analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25–1000 ng/ml (r²≥0.996) with accuracy ≤9.8% and precision ≤13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1300 ng/ml for MPA. This HPLC–MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.     

Liquid chromatographic assay for the antiviral nucleotide analogue tenofovir in plasma using derivatization with chloroacetaldehyde

A sensitive and selective reversed-phase liquid chromatographic assay for tenofovir in human plasma has been developed and validated. Tenofovir was isolated from a 200 microl plasma sample using protein precipitation with trichloroacetic acid. The fluorescent 1,N(6)-etheno derivative is formed at 98 degrees C in the buffered extract with chloroacetaldehyde. This derivative was analysed using gradient ion-pair liquid chromatography and fluorescence detection at 254 nm for excitation and 425 nm for emission. In the evaluated concentration range (20-1000 ng/ml), the intra-day precision was 4% and the inter-day precision was 5-6%. An accuracy of between 97 and 110% was determined. The lower limit of quantification was 20 ng/ml with an inter-day precision of 11%, an intra-day precision of 12% and an accuracy of 103%. The assay is subject to interference from co-administered abacavir. The usefulness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with tenofovir.     

High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment

This paper describes the development of a simple and sensitive analytical method for the quantification of melatonin in human plasma and rabbit serum, using standard analytical equipment and on-line column enrichment without prior extraction, clean-up or derivatization. The analytical procedure was found to be accurate, precise and linear. For human plasma, the accuracy was 101% (range 89–106%), and the mean precision was 5% (range 2–9%) for all concentrations (0, 2, 10, 50 and 200 ng/ml) tested (n=6). The accuracy in rabbit serum was 101% (range 90–112%), and the mean precision was 13% (range 8–19%) for all concentrations (0, 2, 10, 50, 200 and 500 ng/ml) tested (n=6). The retention time of melatonin was about 8 min and the total recoveries were found to be approximately 65 and 85%, respectively, for human plasma and rabbit serum. The limit of detection was found to be lower than 1 ng/ml for human plasma and around 2 ng/ml for rabbit serum. The method is, therefore, found to be suitable for melatonin bioavailability studies in rabbits and presumably also in humans.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg, -Trp, mePhe]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, -Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.     

High-performance liquid chromatographic assay of mepivacaine enantiomers in human plasma in the nanogram per milliliter range

A method enabling quantification of R-(−)- and S-(+)-mepivacaine in human plasma in the low nanogram per milliliter range is described. The procedure involves extraction from plasma with diethyl ether, centrifugation, back-extraction into an acidified aqueous solution, washing with a mixture of pentane and isoamylalcohol, alkalinisation, followed by extraction with a mixture of n-pentane and isoamylalcohol. After evaporation of the organic phase, the residue is redissolved in the mobile phase used for the HPLC analysis, which consists of a 6.8:93.2 (v/v) isopropanol-sodium hydrogenphosphate buffer solution with the pH adjusted to 6.8 using phosphoric acid. The HPLC method has been described previously. Separation of the enantiomers is achieved with an α₁-AGP column and the UV detection wavelength is 210 nm. The minimal detectable concentration is ca. 3 ng/ml and the lower limit of quantification is 5 ng/ml for each enantiomer. For both enantiomers r is >0.9995 over the plasma enantiomeric concentration range of 10.5–1054 ng/ml.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Modified gas chromatographic–mass spectrometric assay for the determination of gacyclidine enantiomers in human plasma

A modified method for the determination of gacyclidine enantiomers in human plasma by GC–MS with selected-ion monitoring using the deuterated derivative of gacyclidine (d₃-gacyclidine) as internal standard was developed. Following a single-step liquid–liquid extraction with hexane, drug enantiomers were separated on a chiral fused-silica capillary column (CP-Chirasil-Dex; Chrompack). The fragment ion, m/z 266, was selected for monitoring d₃-gacyclidine (retention times of 35.2 and 35.6 min for the (+)- and (−)-enantiomer, respectively) whereas the fragment ion, m/z 263, was selected for quantitation of gacyclidine (retention times of 35.4 and 35.9 min for the (+)- and (−)-enantiomer, respectively). The limit of quantitation for each enantiomer was 0.3 ng/ml, using 1 ml of sample, with a relative standard deviation (RSD) <14% and a signal-to-noise ratio of 5. The extraction recovery of both gacyclidine enantiomers from human plasma was about 75%. The calibration curves were linear (r²>0.996) over the working range of 0.312 to 20 ng/ml. Within- and between-day RSD were <9% at 5, 10 and 20 ng/ml, and <16% at 0.312, 0.625, 1.25 and 2.5 ng/ml. Intraday and interday bias were less than 11% for both enantiomers. The chromatographic behavior of d₃-gacyclidine remained satisfactory even after more than 500 injections. Applicability of this specific and stereoselective assay is demonstrated for a clinical pharmacokinetic study with racemic gacyclidine.     

High-performance liquid chromatographic determination of diazepam in plasma of children with severe malaria

A sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection was developed for the quantification of diazepam in small plasma samples from children with severe malaria. The method involves plasma deproteinization with acetonitrile, followed by liquid–liquid extraction with ethyl acetate–n-hexane. Diazepam was eluted at ambient temperatures from a reversed-phase C₁₈ column with an acidic (pH 3.5) aqueous mobile phase (10 mM KH₂PO₄–acetonitrile, 69:31, v/v). Calibration curves in spiked plasma were linear from 10 to 200 ng (r²≥0.99). The limit of detection was 5.0 ng/ml, and relative recoveries at 25 and 180 ng were >87%. Intra- and inter-assay relative standard deviations were <15%. There was no interference from drugs commonly administered to children with severe malaria (phenobarbitone, phenytoin, chloroquine, quinine, sulfadoxine, pyrimethamine, halofantrine, cycloguanil, chlorcycloguanil, acetaminophen and salicylate). This method has been used for monitoring plasma diazepam concentrations in children with seizures associated with severe malaria.     

Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C₈ column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m² CP in a 1-h infusion demonstrated the applicability of the assay.