Determination of pramipexole (U-98,528) in human plasma and urine by high-performance liquid chromatography with electrochemical and ultraviolet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of pramipexole in human plasma and urine. Plasma/urine is made alkaline before pramipexole and BHT-920 (internal standard) are extracted by ethyl ether and back-extracted with a solution that contains heptanesulfonic acid. Separation is achieved by ion-pair chromatography on a Zorbax Rx C₈ column with electrochemical detection at 0.6 V for plasma and ultraviolet detection at 286 nm for urine. The retention times of pramipexole and internal standard are approximately 14.4 and 10.7 min, respectively. The assay is linear in concentration ranges of 50 to 15 000 pg/ml (plasma) and 10 to 10 000 ng/ml (urine). The correlation coefficients are greater than 0.9992 for all curves. For the plasma method, the analysis of pooled quality controls (300, 3000, and 10 000 pg/ml) demonstrates excellent precision with relative standard deviations (R.S.D.) (n=18) of 1.1%, 2.3%, and 6.8%, respectively. For the urine method, quality control pools prepared at 30, 300, and 3000 ng/ml had R.S.D. values (n=18) of 2.9%, 1.7%, and 3.0%, respectively. The plasma and urine controls were stable for more than nine and three months, respectively. The mean recoveries for pramipexole and internal standard from plasma were 97.7% and 98.2%, respectively. The mean recoveries for pramipexole and internal standard from urine were 89.8% and 95.1%, respectively. The method is accurate with all intra-day (n=6) and overall (n=18) mean values for the quality control samples being less than 6.4 and 5.8% from theoretical for plasma and urine, respectively.     

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.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

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.     

Determination of Z-butylidenephthalide in plasma by high-performance liquid chromatography

An HPLC assay is described for the determination of Z-butylidenephthalide (Z-Bdph) in plasma. Plasma samples were cleaned up by extraction with 2% chloroform in n-hexane. Z-Bdph was separated on a normal-phase silica column with a mobile phase of chloroform-n-hexane (1:1). The limit of quantitation with UV detection at 254 nm for Z-Bdph in plasma was 0.01 μg/ml. The recovery of Z-Bdph by organic solvent extraction of plasma was 99.5%. The intra-day and inter-day coefficients of variation and relative errors for Z-Bdph determination in plasma were both less than 10%. The present method was applied to pharmacokinetic studies of Z-Bdph in plasma after intravenous administration to rabbits.     

Determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine using high-performance liquid chromatography with fluorescence detection

A method for the determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine is described. Plasma and urine samples were extracted using 3M Empore extraction disk cartridges in the C₁₈ and MPC (mixed-phase cation-exchange) formats, respectively. The extract was analyzed using HPLC with fluorescence detection (ex 248 nm, em 300 nm), and included a column switching procedure to reduce run-time. The assay was linear in the concentration range 5 to 1000 ng/ml when 1-ml aliquots of plasma and urine were extracted. Recoveries of L-756 423 were greater than 84% over the calibration curve range using the described sample preparation procedures. Intra-day precision and accuracy for this assay was less than 9% RSD and within 7%, respectively. Inter-day variabilities for the plasma (n=17) and urine (n=10) were less than 5% and 3% for low (15 ng/ml) and high (750 ng/ml) quality control samples. Bovine serum albumin (0.5%) was used as an additive to urine to prevent precipitation of L-756 423 during the storage of clinical samples. The assay was used in support of human clinical trials.     

Salivary progesterone for the assessment of the ovarian function in the capuchin monkey (Cebus apella)

Salivary and plasma progesterone were measured in normally cycling (n=10) and castrated (n=4) femaleCebus monkeys (Cebus apella). During the follicular phase, progesterone levels in saliva ranged between 0.05 and 1.40 ng/ml and in the luteal phase they increased to between 0.22 and 4.70 ng/ml. These values represented on average 6.5 and 3.2% of those values measured in plasma, for the follicular and luteal phases, respectively. The regression analysis of the steroid concentrations in both fluids showed a highly significant correlation (r=0.8985,n=180,P<0.0001). Ovariectomized monkeys had consistently low salivary (0.37±0.02 ng/ml) and plasma (4.70±0.25 ng/ml) progesterone, showing a low, but significnat, correlation coefficient (r=0.2592,n=58,P=0.047). The ratio of plasma/salivary progesterone was significantly higher in the luteal phase (31.09±1.65) than in the follicular phase (23.06±2.26) and in castrated monkeys (16.00±1.38). The free fraction of progesterone constituted 5.3±0.2% of the total plasma progesterone during the follicular phase and 3.3±0.1% during the luteal phase. Ovariectomized monkeys showed a significantly higher percentage of free progesterone in plasma (7.7±0.1%). In contrast, free progesterone made up 64.4 and 70.9% of the total salivary progesterone for the follicular and luteal phases, respectively. The proportion of free progesterone in castrated animals was within the range observed in cycling animals. We suggest that the levels of progesterone in the saliva of capuchin monkey follow a pattern similar to that for plasma progesterone, reflecting the free steroid fraction. Thus, the measurement of such steroid in saliva may offer a valuable alternative to plasma determinations for the assessment of the ovarian function inCebus and probably other New World monkey species.     

High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

Simultaneous determination of all-trans-, 13-cis-, 9-cis-retinoic acid and their 4-oxo-metabolites in plasma by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatographic technique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and their corresponding 4-oxometabolites, in plasma. The method involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C₁₈ steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient composition was (only the percentages of solvent B are mentioned): I, 25% solvent B at the time of injection; II, 12% solvent B at 11 min until 30 min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detection was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the range of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ml for each of the compounds. The HPLC method was applied to plasma specimens collected from animals receiving single dose administrations of all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid.     

Influence of season and body condition on plasma volume levels of white‐tailed deer,Odocoileus virginianus

Abstract

Changes in the circulating plasma volume were monitored for twelve consecutive months in five white‐tailed deer (Odocoileus virginianus) maintained outdoors in a 2.4‐ha enclosure in northern New York stale. The mean annual relative plasma volume of the two male white‐tailed deer (43.6 ± 1.0 ml/kg) was not significantly different from that of three females (45.7 ± 0.7 ml/kg). Mean annual absolute plasma volume, however, was significantly higher in males (2588 ± 90.7 ml) than in females (2092 ± 79.1 ml), mainly because of the greater body weights of males in late summer and early fall. Both sexes had a marked seasonality in the level of relative and absolute plasma volume regulation. Relative plasma volumes were lowest in February‐March and highest in May‐June and again in October, while absolute plasma volumes were lowest in late winter to early spring, when body weights in the annual cycle were lowest, and volumes were highest in mid‐fall, at the annual peak of body condition. Males had a greater excursion about their annual mean relative plasma volumes (‐16 to 13%) and absolute plasma volumes (‐23 to 32%) than did females (respectively, ‐11 to 9% and ‐13 to 19%). Variations in the level of plasma volume regulated are related to the annual cycle of environmental conditions and changes in body and physiological condition of white‐tailed deer.     

Quantitative determination of clonazepam in plasma by gas chromatography-negative ion chemical ionization mass spectrometry

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the quantitative analysis of clonazepam in human plasma is described. Clonazepam (M_r = 315) was derivatized by N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. A pre-conditioning procedure involving injection of a silyl-8 and ethyl acetate extraction solution from plasma reduces the interaction between clonazepam-TMS and the analytical system. The routine limit of quantification was set to be 0.25 ng/ml with an injection volume of 2 μl and a sample volume of 1 ml. The signal-to-noise ratio was greater than five. The detection limit for clonazepam can reach 0.1 ng/ml. The isotope clonazepam-d₅ was used as the internal standard.     

Influence of Exogenous Interleukin-2 Concentration on the Isolation of Human Immunodeficiency Virus

A significant increase (P=0.015) in the HIV isolation rate from plasma samples was achieved by use of 10 U/ml exogenous interleukin-2 compared to 20 U/ml. The sensitivity rose from 0% to 29% on patients negative for p24 core antigen (P=0.031) and from 71% to 86% in patients positive for p24 core antigen in plasma (P>0.05). Titration of infectious HIV revealed that 10 U/ml interleukin-2 is the optimal concentration to isolate low numbers of infectious particles of HIV.     

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.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine–glucuronide in plasma by liquid chromatography–tandem mass spectrometry

For the first time, an LC–MS–MS method has been developed for the simultaneous analysis of buprenorphine (BUP), norbuprenorphine (NBUP), and buprenorphine–glucuronide (BUPG) in plasma. Analytes were isolated from plasma by C₁₈ SPE and separated by gradient RP-LC. Electrospray ionization and MS–MS analyses were carried out using a PE-Sciex API-3000 tandem mass spectrometer. The m/z 644→m/z 468 transition was monitored for BUPG, whereas for BUP, BUP-d₄, NBUP, and NBUP-d₃ it was necessary to monitor the surviving parent ions in order to achieve the required sensitivity. The method exhibited good linearity from 0.1 to 50 ng/ml (r²≥0.998). Extraction recovery was higher than 77% for BUPG and higher than 88% for both BUP and NBUP. The LOQ was established at 0.1 ng/ml for the three analytes. The method was validated on plasma samples collected in a controlled intravenous and sublingual buprenorphine administration study. Norbuprenorphine–glucuronide was also tentatively detected in plasma by monitoring the m/z 590→m/z 414 transition.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

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.     

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

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Measurement of hyperforin a constituent of St. John’s wort in plasma by high-performance liquid chromatography

Hyperforin is a constituent of Hypericum perforatum extracts (St. John’s wort, H. perforatum), which have antidepressant action. Hyperforin was extracted from plasma utilising a solid-phase extraction procedure. Chromatography was performed by isocratic reversed-phase high-performance liquid chromatography with UV end-point detection. The calibration curve was linear over the range 0.15–3 μg per ml of plasma. The sensitivity for hyperforin was 4.5 ng on-column. Mean inter- and intra-assay relative standard deviations over the range of the standard curve were less than 5%. The absolute recovery for hyperforin averaged 97.8%.     

Determination of pramipexole (U-98,528) in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A highly sensitive and selective HPLC-MS-MS method was developed for the determination of pramipexole in human plasma. The analytes, pramipexole and BHT-920 (internal standard), were extracted from plasma at basic pH with methyl tert.-butyl ether (MTBE). MTBE was evaporated to dryness and reconstituted in 100 μl of (95:5) methanol-water. Chromatographic separation was achieved on a Zorbax SB-CN column with a mobile phase of (15:5:80) water-0.1 M ammonium acetate—methanol. The analytes were detected utilizing HPLC in conjunction with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MSM). The assay was linear in the concentration ranges of 50 to 5000 pg/ml. The analysis of pooled quality controls (150, 750, and 3000 pg/ml) demonstrated excellent precision with relative standard deviations (R.S.D.) (n=18) of 7.2%, 5.3% and 5.2%, respectively. The method is accurate with all intra-day (n=6) and overall (n=18) mean values being less than 11.7% from theoretical.