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.     

Purge-and-trap gas chromatographic determination of styrene in urine and blood: Application to exposed workers

A simple purge-and-trap gas chromatographic method with flame ionization detection was developed for the determination of styrene in urine and blood. Styrene present in a 5 ml sample at room temperature was swept by helium at 40 ml/min for 11 min, trapped on a Tenax trap, desorbed by heating, cryofocused, and injected by flash heating into a DB-5 capillary GC column. The oven temperature program was from 80°C, held for 8 min, to 120°C at 5°C/min, and then held for 2 min. The detector temperature was 250°C. The calibration curves were linear in the range of 2.5–15 ppb styrene in urine and 25–150 ppb in blood. The detection limits calculated were 0.4 μg/l in urine and 0.6 μg/l in blood. The coefficients of variations within the day and day-to-day were 3 and 3.1%, respectively, for 2.5 ppb of styrene in urine, and 1 and 1.6% for 25 ppb of styrene in blood. The results obtained from samples taken from workers exposed to styrene were reported.     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of -arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [¹⁵N]nitrite and [¹⁵N]nitrate were used as internal standards. Endogenous nitrite and [¹⁵N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [¹⁵N]nitrate they were reduced to nitrite and [¹⁵N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [¹⁵N]nitrite ([¹⁵N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Improved assay for the quantification of 11-dehydrothromboxane B2 by gas chromatography—mass spectrometry

Endogenous thromboxane production is best assessed by the measurement of its excreted metabolites, of which 11-dehydrothromboxane B₂ (11-dehydro-TxB₂) is most abundant. Gas chromatographic—mass spectrometric assays have been developed for this compound but suffer from the presence of co-eluting impurities which make the measurement of 11-dehydro-TxB₂ difficult. Furthermore, these assays are often time-consuming. We now report a modified assay for the measurement of this compound employing gas chromatography—mass spectrometry which alleviates the problem of co-eluting impurities primarily through modification of extraction and chromatographic methods. Furthermore, the time to complete the assay is significantly shortened. It is adaptable to both urine and plasma. Precision of the assay is ± 7% and accuracy is 90%. The lower limit of sensitivity in urine is approximately 20 pg/mg creatinine. Normal levels of urinary excretion of this metabolite were found to be 370 ± 137 pg/mg creatinine (mean ± 1 S.D.) and normal plasma levels were found to be 1.5 ± 0.4 pg/ml (mean ± 1 S.D.). Urinary excretion of 11-dehydro-TxB₂ is markedly altered in situations associated with abnormalities in thromboxane generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous thromboxane production and to further explore the role of this compound in human disease.     

Small blood volumes from children for quantitative sotalol determination using high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method using fluorescence detection has been developed for sotalol determination in small plasma samples of children and newborns with limited blood volume. In sample sizes of 100 μl of plasma, sotalol was extracted using an internal standard and solid-phase extraction columns. Chromatographic separation was performed on a Spherisorb C₆ column of 150×4.6 mm I.D. and 5 μm particle size at ambient temperature. The mobile phase consisted of acetonitrile–15 mM potassium phosphate buffer (pH 3.0) (70:30, v/v). The excitation wavelength was set at 235 nm, emission at 300 nm. The flow-rate was 1 ml/min. Sotalol and the internal standard atenolol showed recoveries of 107±8.9 and 97±8.1%, respectively. The linearity range for sotalol was between 0.07 and 5.75 μg/ml, the limit of quantitation 0.09 μg/ml. Precision values expressed as percent relative standard deviation of intra-assay varied between 0.6 and 13.6%, that of inter-assay between 2.4 and 14.4%. Accuracy varied between 86.1 and 109.8% (intra-assay) and 95.4 and 103.3% (inter-assay). Other clinically used antiarrhythmic drugs did not interfere. As an application of the assay, sotalol plasma concentrations in a 6-year-old child with supraventricular tachycardia treated with oral sotalol (3.2 mg/kg per day) are reported.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

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.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C₁₈ column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 ± 3.7% (n = 45) and 100 ± 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 μmol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.     

Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10–15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.     

A high-performance liquid chromatographic method for the determination of pseudouridine and uric acid in native human urine and ultrafiltrated serum

A simple and reliable HPLC method for quantitative determination of pseudouridine and uric acid in human urine and serum using a cation-exchange resin is described. This method is straightforward (12 runs of urine samples per day since the sample is only diluted into buffer and then chromatographed), sensitive, and highly reproducible. The column is stable over long periods (3 months of uninterrupted use at a time; it is thereafter easily restored to the original state). Mean excretion values for pseudouridine (in μmol/mmol creatinine) are 26.4 ± 3.1 (17 female adults), 23.8 ± 2.5 (12 male adults), 164.7 ± 32.2 (37 male preterm infants); mean values for uric acid (μmol/mmol creatinine) are, respectively, 310.3 ± 90.5, 278.2 ± 56.1, and 1108 ± 314. Human serum is deproteinized by pressure ultrafiltration in microcollodion bags with a nominal exclusion molecular weight of 12,400 and then put directly onto the HPLC column. The complete procedure takes 4 h.     

Determination of tetrahydropapaveroline in the urine of Parkinsonian patients receiving l-dopa—carbidopa (Sinemet) therapy by high-performance liquid chromatography

Tetrahydropapaveroline (THP) concentrations were measured in the urine of Parkinsonian patients receiving l-dopa—carbidopa (Sinemet) therapy, using a method that employs a separation scheme that selectively isolates THP from urine and utilizes the Pictet—Spengler condensation of THP with formaldehyde combined with high-performance liquid chromatography for identification and determination. The mean (± S.D.) recoveries of THP from normal urine with 0.2 pmol/ml added and from Parkinsonian patients' urines with 0.5 pmol/ml added were 48.6 ± 5.7 and 44.6 ± 3.1%, respectively. Three Parkinsonian patients who were receiving either 250, 750 or 1000 mg of l-dopa (as Sinemet) daily had 24-h urinary THP excretion levels of 989, 1017 and 1600 pmol, respectively.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients

CPT-11 {I; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin} is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C₁₈) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C₁₈ reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml–10 μg/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5–1000 ng/ml) was 13.0% (range 4.9–19.4%) for I and 12.8% (6.7–19.1%) for II; the between-day R.S.D. (5–10 000 ng/ml was 7.9% (5.4–17.5%) for I and 9.7% (3.5–15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m² I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 ± 285 ng/ml (mean ± standard error of the mean). Plasma decay was triphasic with half-lives α, β and γ of 5.4 ± 1.8 min, 2.5 ± 0.5 h and 20.2 ± 4.6 h, respectively. The volume of distribution at steady state was 105 ± 15 l/m², and the total body clearance was 12.5 ± 1.9 l/h · m². The maximum concentrations of the active metabolite II reached 36 ± 11 ng/ml.     

Sensitive method for the determination of bisphenol-A in serum using two systems of high-performance liquid chromatography

The aim of this study was to establish an easy and accurate method for the determination of bisphenol-A (BPA) in the body liquid such as serum and urine. Two high-performance liquid chromatography (HPLC) systems, HPLC with electrochemical detector (ED), and HPLC with mass spectrometry (MS) using electrospray ionization (ESI) interface were used for the assay in the serum samples prepared with solid-phase extraction method. Water or EtOH at a concentration below 50% was suitable for the extraction of BPA from serum. The limit of detection of BPA was 0.2 ng ml⁻¹ for the HPLC-ED method and 0.1 ng ml⁻¹ for HPLC–MS. There was a good correlation between the data obtained by the two HPLC systems. BPA concentrations in healthy human serum were low (0–1.6 ng ml⁻¹). From various commercial fetal bovine serum and sheep plasma, however, significant amounts of BPA were detected. Since no BPA was detected from sheep plasma immediately after collection, the high amounts of BPA were considered to be caused by the handling of blood during the preparation of the products after blood collection. In vitro study showed that the amount of BPA leached from polycarbonate tube into sheep plasma were 40 times larger than those into water and the leached amount of BPA depended on the temperature (37°C>20°C>5°C).     

Determination of diphenylpyraline in plasma and urine by high-performance liquid chromatography

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

Evaluation of genotoxic effects of Apitol (cymiazole hydrochloride) in vitro by measurement of sister chromatid exchange

Apitol^®, with cymiazole hydrochloride as the active ingredient, is used in bee-keeping against the ectoparasitic mite Varroa destructor. The preparation was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister chromatid exchange, the mitotic index and the cell proliferation index were determined for three experimental concentrations of Apitol^® (0.001, 0.01 and 0.1 mg/ml). All concentrations significantly (p < 0.001) increased the mitotic index (MI = 7.35 ± 0.18%, 8.31 ± 0.20% and 12.33 ± 0.25%, respectively), the proliferative index (PI = 1.83 ± 0.01, 1.84 ± 0.01 and 1.88 ± 0.02, respectively) and the frequency of sister chromatid exchange (SCE = 8.19 ± 1.81, 8.78 ± 1.80 and 13.46 ± 1.88, respectively), suggesting that cymiazole hydrochloride has genotoxic potential.