Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

High-performance liquid chromatographic procedures for the quantitative determination of paclitaxel (Taxol) in human urine

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 μM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 μg/ml drug concentration. The lower limit of quantitation is 0.01 μg/ml for SPE and 0.25 μg/ml for LLE. Stability data of paclitaxel in human urine are also presented.     

Determination of lycopene in tissues and plasma of rats by normal-phase high-performance liquid chromatography with photometric detection

An analytical method for the determination of lycopene in tissues and plasma of rats is described. The method was validated for the determination of lycopene in liver and plasma with respect to selectivity, linearity, accuracy, recovery and precision. Following precipitation of proteins with water–ethanol plasma was extracted with hexane; tissues were extracted with acetone followed by precipitation of proteins with water–ethanol and extraction of lycopene with hexane. Separation and quantification of geometrical isomers of lycopene was achieved by normal-phase HPLC with UV/VIS detection at 471 nm. The method proved to be selective and specific for lycopene in plasma and liver. Detector response was linear in the range from 2 ng/g to 10 μg/g liver and 0.5 ng/ml to 2 μg/ml plasma, respectively. Average recoveries ranged from 96 to 101% in spiked liver samples and from 91 to 94% in spiked plasma samples. Intra-day variability (C.V.) was ≤6% and ≤5% in liver and plasma, respectively. Inter-day precision was ≤9% for liver samples and ≤6% for plasma samples. The procedures were successfully applied to the sample analysis of pharmacokinetic and metabolism studies.     

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.     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Simple high-performance liquid chromatographic method for the quantitation of 5-fluorouracil in human plasma

A simple, rapid, specific and sensitive high-performance liquid chromatography method has been developed for quantitation of 5-fluorouracil (5-FU) in human plasma. The method involves deproteinization of a small sample volume of plasma (150 μl) followed by HPLC on a cation-exchange resin column, Aminex HPX-87H (300×7.8 mm I.D.), preceded by a similar guard cartridge with UV detection at 265 nm. This method allows a good separation of 5-FU with a retention time of 24 min and a detection limit at 25 ng/ml. The calibration curve was linear from 25 to 2000 ng/ml. The coefficient of variation was ≤4.4% for within-day reproducibility and ≤9.5% for day-to-day reproducibility.     

Development and validation of a high-performance liquid chromatographic assay using solid-phase extraction for the novel antitumor agent pancratistatin in human plasma

The stability of the experimental anti-tumour agent pancratistatin in human plasma has been investigated. A solid-phase extraction technique and an HPLC assay with external standards have been developed and validated. Extraction was performed using C₁₈ cartridges and HPLC, analysis was performed on a 15 cm Hypersil BDS column using isocratic elution with 13% acetonitrile and aqueous solution of 1% (w/v) acetic acid. The lower limit of quantification for pancratistatin in 5% DMF–95% water was found to be 0.58 ng/ml (±10.58%) and 2.3 ng/ml (±9.2%) following extraction from human plasma. Mean recovery of 89.4% (±4.73%) was obtained over the concentration range 0.0023–9.45 μg/ml for a five day validation study. Pancratistatin was stable at room temperature in light or dark for at least 15 days, in the refrigerator at 4°C for at least 16 days and in the freezer at −20°C or −80°C for at least 28 days. Under all conditions monitored, % recovery of pancratistatin from human plasma was greater than 95% and no evidence of degradation had occurred. There also was no loss of pancratistatin after three cycles of freezing and thawing.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Selective extraction of quercetrin in vegetable drugs and urine by off-line coupling of boronic acid affinity chromatography and high-performance liquid chromatography

Quercetrin, quercetin and chlorogenic acid were measured in urine or in drugs by combination of boronic acid affinity chromatography and HPLC. Simple reversed-phase HPLC with UV detection was used to determine quercetrin in five different Solidago virgaurea drugs. For determination of quercetrin in human urine immobilized boronic acid was applied for sample pretreatment. This procedure leads to a determination limit of 0.01 μg/ml with a recovery rate of 95.3%. The first results using this method for quercetrin pharmacokinetics are presented.     

High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva

A sensitive and specific high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of minocycline in human plasma and parotid saliva samples. Samples were extracted using an Oasis™ HLB cartridge and were injected into a C₈ Nucleosil column. The HPLC eluent contained acetonitrile–methanol–distilled water–0.1% trifluoroacetic acid (25:2:72.9:0.1, v/v). Demeclocycline was used as internal standard. The assay showed linearity in the tested range of 0.1–25 μg/ml. The limit of quantitation was 100 ng/ml. Recovery from plasma or parotid saliva averaged 95%. Precision expressed as %CV was in the range 0.2–17% (limit of quantitation). Accuracy ranged from 93 to 111%. In the two matrices studied at 20 and 4°C, rapid degradation of the drug occurred. Frozen at −30°C, this drug was stable for at least 2 months, the percent recovery averaged 90%. The method’s ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.     

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.     

Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative

We established a method for the detection of free and total (free and bound) malondialdehyde (MDA) in human plasma samples after derivatisation with 2,4-dinitrophenylhydrazine (DNPH). Free MDA was prepared by perchloric acid deproteinisation whereas an alkaline hydrolysation step for 30 min at 60°C was introduced prior to protein precipitation for the determination of total MDA. Derivatisation was accomplished in 10 min at room temperature subsequently chromatographed by HPLC on a reversed-phase 3 μm C₁₈ column with UV detection (310 nm). The detection limit was 25 pmol/ml for free and 0.3 nmol/ml for total MDA. The recovery of MDA added to different human plasma samples was 93.6% (n=11; RSD 7.1%) for the hydrolysation procedure. In samples from 12 healthy volunteers who underwent a hypoxic treatment (13% O₂ for 6 h) we estimated a baseline value of total MDA of 2.16 nmol/ml (SD 0.29) (ambient air) with a significant increase to 2.92 (nmol/ml, SD 0.57; P=0.01) after the end of this physiological oxidative stress challenge. Plasma values of free MDA in these samples were close to our detection limit. The presented technique can easily performed with an isocratic HPLC apparatus and provides highly specific results for MDA as do sophisticated GC–MS methods.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C₂ ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10–2500 ng/ml in plasma with coefficients of variation ≤ 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.     

Rapid and Simultaneous Quantification of Levetiracetam and Its Carboxylic Metabolite in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

A simple liquid chromatography tandem mass spectrometry method was developed and validated according to the guidelines of the US Food and Drug Administration and the European Medicines Agency for a simultaneous quantification of levetiracetam (LEV) and its metabolite, UCB L057 in the plasma of patients. A 0.050 mL plasma sample was prepared by a simple and direct protein precipitation with 0.450 mL acetonitrile (ACN) containing 1 µg/mL of internal standard (IS, diphenhydramine), then vortex mixed and centrifuged. A 0.100 mL of the clear supernatant was diluted with 0.400 mL water and well mixed. A 0.010 mL of the resultant solution was injected into an Agilent Zorbax SB-C18 (2.1 mm×100 mm, 3.5 µm) column with an isocratic elution at 0.5 mL/min using a mixture of 0.1% formic acid in water and ACN (40∶60 v/v). Detection was performed using an AB Sciex API 3000 triple quadrupole mass spectrometer, equipped with a Turbo Ion Spray source, operating in a positive mode: LEV at transition 171.1>154.1, UCB L057 at 172.5>126.1, and IS at 256.3>167.3; with an assay run time of 2 minutes. The lower limit of quantification (LLOQ) for both LEV and UCB L057 was validated at 0.5 µg/mL, while their lower limit of detection (LOD) was 0.25 µg/mL. The calibration curves were linear between 0.5 and 100 µg/mL for both analytes. The inaccuracy and imprecision of both intra-assay and inter-assay were less than 10%. Matrix effects were consistent between sources of plasma and the recoveries of all compounds were between 100% and 110%. Stability was established under various storage and processing conditions. The carryovers from both LEV and UCB L057 were less than 6% of the LLOQ and 0.13% of the IS. This assay method has been successfully applied to a population pharmacokinetic study of LEV in patients with epilepsy.     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C₁₈ cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g (r²=0.9998) and for flumequine in the range of 0.3–50 μg/g (r²=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.     

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.