High-performance Liquid chromatography-UV Method Research Articles

Micelle to cyclodextrin stacking in open-tubular liquid chromatography using capillaries coated with surfactant admicelles.

In-line sample concentration by micelle to cyclodextrin stacking (MCDS) in open-tubular liquid chromatography (OT-LC) with UV detection is described. OT-LC of two sets of analytes (small-molecule drugs and neutral alkenylbenzenes) was by the use of a fused-silica capillary that was coated with admicelles of didodecyldimethyl ammonium bromide (DDAB). These admicelles acted as a stationary chromatographic pseudophase. The mobile phase was 25mM sodium tetraborate in 10% methanol, pH 9.2. MCDS was by long pressure injection of samples prepared in 10mM hexadecyltrimethyl ammonium bromide (CTAB) in 25mM sodium tetraborate, pH 9.2 (buffer), followed by injection of 50mM α-CD in buffer (CD solution). Stacking was by application of voltage at -20kV prior to pressure-driven OT-LC. The factors that influenced MCDS such as type and concentration of CD, concentration of CTAB in the sample, injection time ratio of the sample and the CD solution and stacking time were studied. Under optimised conditions, sensitivity enhancement factors (SEFs) were between 19 and 23, linear ranges were between 0.5 and 10µg/mL with r2 > 0.99 and inter-day/intra-day repeatability in retention time and peak area were ≤5.6% for the model small-molecule drugs. Application to real samples was by the determination of potentially toxic alkenylbenzenes (SEFs = 10 to 12) in basil-leaf and whole-clove extracts. The assay results were comparable to those obtained from an in-house high-performance liquid chromatography-UV method.

Pharmacokinetics of furosemide in goats following intravenous, intramuscular, and subcutaneous administrations.

Furosemide, a loop diuretic drug, is recommended for use in cases of edema, ascites, congestive heart failure, toxicosis, and acute renal failure in goats. However, its pharmacokinetics and bioavailability have not been reported yet in this species. The aim of this study was to determine the pharmacokinetics and bioavailability of furosemide in goats following intravenous (IV), intramuscular (IM), and subcutaneous (SC) administrations at a dose of 2.5mg/kg. Six clinically healthy goats received furosemide by each route in a three-way crossover pharmacokinetic design with a 15-day washout period between administrations. The plasma concentrations of furosemide were determined using the high-performance liquid chromatography-UV method and analyzed by non-compartmental analysis. The elimination half-life following IV, IM, and SC administration was 0.71 (0.67-0.76)h, 0.69 (0.61-0.74)h, and 0.70 (0.67-0.79)h, respectively. The volume of distribution at steady state and total clearance for the IV route were 0.17 (0.16-0.19)L/kg and 0.30 (0.27-0.33)L/h/kg, respectively. The peak plasma concentrations of furosemide following IM and SC administrations were 11.19 (10.33-11.95) and 6.49 (5.92-7.00)μg/ml at 0.23 (0.16-0.25) and 0.39 (0.33-0.42)h, respectively. The bioavailability was 109.84 (104.92-116.99)% and 70.80 (55.77-86.67)% for the IM and SC routes, respectively. The pharmacokinetics of furosemide following the IV, IM, and SC administrations in goats demonstrated significant differences, which may have clinical and toxicological implications requiring further investigations.

Impact of renal function on hydroxyurea exposure in sickle-cell disease patients.

This prospective study aimed to develop a population pharmacokinetics (PK) model of hydroxyurea (HU) in patients with sickle cell disease. This model can be used to determine the impact of glomerular filtration rate (GFR) on HU kinetics. We included 30 patients. They underwent HU pharmacokinetics analyses of plasma and urine. Six underwent PK analyses in 2 periods with and without angiotensin-converting enzyme inhibitor. HU was assayed with a validated high-performance liquid chromatography-UV method. Noncompartmental PK analysis was conducted and a population PK model built with Monolix. This model was validated externally on another 56 patients. HU PK was simulated as a function of GFR. The HU PK model was constructed as a 2-compartment model with first-order absorption and elimination. The quality criteria were good, including for external validation. We found that estimated GFR (eGFR) and body weight affected HU PK, with lower eGFR or body weight associated with a higher HU area under the curve. We recommend the monitoring of HU through eGFR and body weight, which together account for 47% of its variability. Urinary HU fractions and renal clearance were higher in the glomerular hyperfiltration group and lower in the moderate chronic kidney disease group, respectively. No differences in nonrenal HU clearance were observed. Estimated GFR has an impact on the kinetics of hydroxyurea, and HU dose should be adapted accordingly. Angiotensin-converting enzyme inhibitor seems to have minor effect on HU PK in adults with sickle cell disease.

HPLC method for Residual 2-Bromoethylamine Hydrobromide in Hemoglobin-based Oxygen Carriers Derived with 4-Methoxybenzenesulfonyl Chloride

A stable high-performance liquid chromatography-UV (HPLC) method was developed for the separation and quantification of residual 2-bromoethylamine hydrobromide (BEA) in hemoglobin-based oxygen carriers (HBOCs). Residual BEA was derived by 4-Methoxybenzenesulfonyl chloride (MOBS-Cl) at 20 °C for 5 min in a pyridine solution. With this organic reaction system, MOBS-Cl hydrolysis in aqueous phase can be avoided in the process of derivatization, and glycine (Gly), a protein stabilizer, does not dissolve and extract from the organic reaction system, thus avoiding the serious interference of Gly on BEA determination. After derivatization, the derivative product was analyzed by HPLC with a mobile phase of 35% acetonitrile and 65% 0.01 mol/L ammonium acetate solution (v:v) and detected at 240 nm. The linear range was 0.41–40.98 μg/mL, with a correlation coefficient R2 of >0.9976. Excellent method reproducibility was observed by intra- and inter-day precision, with a relative standard deviation of <3%. The detection limit (S/N = 3) was found to be <0.02 μg/mL. This method can be used for routine determination of residual 2-bromoethylamine hydrobromide in hemoglobin-based oxygen carriers and other biological medicines. The proposed method also provides a new strategy for the separation and determination of other amino compounds which can dissolve in organic phase.

Pharmacokinetics of ceftriaxone following single ascending intravenous doses in sheep

The objective of this study was to evaluate the pharmacokinetics of CTX following intravenous administration of ascending doses in sheep. In this study, six clinically healthy Akkaraman sheep (2.4 ± 0.4 years and 50 ± 3 kg of body weight) were used. CTX was administered intravenously to each sheep at 20, 40, and 80 mg/kg doses in a crossover design with a 15-day washout period. Plasma concentrations of CTX were measured using the high-performance liquid chromatography-UV method. Pharmacokinetic parameters were calculated by non-compartmental analysis. CTX was well tolerated following administration at 20, 40, and 80 mg/kg doses. The elimination half-life following administration of 40 and 80 mg/kg doses were significantly longer than that of 20 mg/kg dose (P < 0.05). The volume of distribution at steady state was similar among the groups (P > 0.05). When compared to 20 mg/kg, dose-normalized AUC0–∞ at the 80 mg/kg dose significantly increased (P < 0.05). The relation between dose and AUC0–∞ was linear. Our study showed that CTX can be used at 12-h intervals for 20, 40, and 80 mg/kg doses to maintain T > minimum inhibitory concentration (MIC) of >40% for the treatment of infections caused by bacteria with MIC values ≤2, ≤4, and ≤16 μg/mL, respectively. This information may be helpful in adjusting the dosage regimen, but there is a need for future work.

Rapid Screening for 15 Sulfonamide Residues in Foods of Animal Origin by High-Performance Liquid Chromatography-UV Method.

A rapid and simple high-performance liquid chromatography-ultraviolet method was developed for the separation and quantification of 15 sulfonamides (SAs) in foods of animal origin without the need of clean-up procedure. A mixture of acetonitrile-formic acid-ammonium acetate-water was used as the mobile phase to separate 15 SAs on a C18 column with gradient. The selected SAs were separated completely from the matrix mixture based on different retention behaviors at different concentration of acetonitrile. The effects of the additive of formic acid and ammonium acetate in mobile phases on the separation of SAs were also investigated. The additive can greatly improve the resolution between SAs and impurities, so that the SAs can be quantified directly under the optimized chromatographic condition the sample preparation which does not need extra sample clean-up procedure. Complete baseline separation of 15 SAs was achieved in <40 min, the linear range is 0.01-130 μg/mL with a correlation coefficient R2-value > 0.999. Excellent method reproducibility was found by intra- and inter-day precisions with the relative standard deviation <9.5%. The detection limit was <11.0 ng/mL and it can be used for routine screening of the SA residues in foods of animal origin.

Impact of Infection Status and Cyclosporine on Voriconazole Pharmacokinetics in an Experimental Model of Cerebral Scedosporiosis.

Cerebral Scedosporium infections usually occur in lung transplant recipients as well as in immunocompetent patients in the context of near drowning. Voriconazole is the first-line treatment. The diffusion of voriconazole through the blood-brain barrier in the context of cerebral infection and cyclosporine administration is crucial and remains a matter of debate. To address this issue, the pharmacokinetics of voriconazole was assessed in the plasma, cerebrospinal fluid (CSF), and brain in an experimental model of cerebral scedosporiosis in rats receiving or not receiving cyclosporine. A single dose of voriconazole (30 mg/kg, i.v.) was administered to six groups of rats randomized according to the infection status and the cyclosporine dosing regimen (no cyclosporine, a single dose, or three doses; 15 mg/kg each). Voriconazole concentrations in plasma, CSF, and brain samples were quantified using ultra-performance liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography UV methods and were documented up to 48 hours after administration. Pharmacokinetic parameters were estimated using a noncompartmental approach. Voriconazole pharmacokinetic profiles were similar for plasma, CSF, and brain in all groups studied. The voriconazole Cmax and area under the curve (AUC) (AUC0 ≥ 48 hours) values were significantly higher in plasma than in CSF [CSF/plasma ratio, median (range) = 0.5 (0.39-0.55) for AUC0 ≥ 48 hours and 0.47 (0.35 and 0.75) for Cmax]. Cyclosporine administration was significantly associated with an increase in voriconazole exposure in the plasma, CSF, and brain. In the plasma, but not in the brain, an interaction between the infection and cyclosporine administration reduced the positive impact of cyclosporine on voriconazole exposure. Together, these results emphasize the impact of cyclosporine on brain voriconazole exposure.

Hydroxymethylfurfural in fruit puree and juice: preconcentration and determination using microextraction method coupled with high-performance liquid chromatography and optimization by Box–Behnken design

In the present study, a sensitive and rapid method for separation and determination of hydroxymethylfurfural (HMF) in fruit puree and juices was proposed. Dispersive liquid–liquid microextraction (DLLME) coupled with high-performance liquid chromatography (HPLC) was used for extraction and quantitative determination of HMF in fruit puree and juices. The effective parameters such as the type and volume of extraction and dispersive solvents, pH and salt amount (NaCl) were studied and optimized with the aid of response surface methodology based on Box–Behnken design to obtain the best condition for HMF extraction. At the optimized conditions, parameter values were 60 µL extracting solvent, 600 µL dispersive solvent, 2 g NaCl and pH 5. Repeatability of the method, described as the relative standard deviation, was 3.1% (n = 6) and the recovery was 98.4%. The limit of detection and limit of quantitation were 1.47 and 5.28 µg L−1, respectively. The merit figures of DLLME–HPLC–UV method showed that the proposed method can be noticed as a new, fast and good alternative method for investigation of HMF in various fruit puree and juice samples.

Analytical method validation and monitoring of diacetyl in liquors from Korean market.

Diacetyl is a natural fermentation by-product and is an important flavor component of certain liquors. This paper aims to validate the high performance liquid chromatography (HPLC) method based on derivatization with 1,2-diaminobenzene for diacetyl quantification in liquor samples. A limit of quantitation of 0.039mg/L was obtained. Coefficient regression (R2) of calibration curve for the HPLC-UV method exceeded 0.999, showing adequate linearity on the standard curve. Relative standard deviation values obtained from intraday and interday analysis for precision were 2.5 and 4.1%, respectively. Using the validated method, the contents of diacetyl in liquors (12 types, 389 samples) distributed throughout Korea were monitored. The average diacetyl content of all analyzed liquor samples ranged from trace amounts to 3.655mg/L (microbrewery beer). The highest average diacetyl content was found in fruit wines (0.432mg/L), followed by red wine (0.320mg/L) and general distilled spirits (0.249mg/L). In takju and yakju, no distinctive effect of sterilization on diacetyl content was found.

A Simple High-Performance Liquid Chromatography for Determining Lapatinib and Erlotinib in Human Plasma.

Lapatinib and erlotinib are used for cancer treatment, showing large interindividual variability. Therapeutic drug monitoring may be useful for assessing the clinical outcomes and adverse events. A simple high-performance liquid chromatography UV method was developed for the determination of lapatinib and erlotinib in human plasma. An aliquot of plasma sample spiked with internal standard was treated with acetonitrile to precipitate the proteins. Lapatinib and erlotinib were separated on an octadecylsilyl silica gel column using a mobile phase consisting of acetonitrile, methanol, water, and trifluoroacetic acid (26:26:48:0.1) pumped at a flow rate of 1.0 mL/min. The detection wavelength was set at 316 nm. The calibration curves for lapatinib and erlotinib were linear (r = 0.9999) in the range of 0.125-8.00 mcg/mL. The extraction recoveries for both lapatinib and erlotinib at the plasma concentration of 0.125-8.00 mcg/mL were higher than 89.9% with coefficients of variation less than 3.5%. The coefficients of variation for intraday and interday assays of lapatinib and erlotinib were less than 5.1% and 6.1%, respectively. The present method can be used for blood concentration monitoring for lapatinib or erlotinib in exactly the same conditions.

P.2.b.028 - The importance of screening for depressive symptoms in patients with diabetes mellitus type 2 in primary health care

Because of its systemic action, fluconazole 1026 is prescribed for a variety of fungal infections. However, therapeutic failure might result when a patient is switched between an innovator drug and a nonbioequivalent generic formulation. Pharmacokinetic (PK) studies investigating the bioequivalence of generic and innovator drugs can minimize such risks.The aim of this study was to compare the 1026 PK profiles and relative bioavailabilities of 2 oral formulations of fluconazole: Diflucan® (reference; Pfizer Corporation Austria GmbH, Wien, Austria) and Funzol® (test; Bosnalijek d.d., Pharmaceutical and Chemical Industry, Sarajevo, Bosnia and Herzegovina), both prepared as capsules containing 150 mg of active drug.A single oral dose of fluconazole waMethods:026 given under fasting conditions to healthy, white volunteers aged 18 to 55 years in this open-label, randomized, crossover study. A 3-week washout period was applied between each of the 2 doses. Serum samples were obtained before dosing and at various time points after dosing up to 144 hours and were analyzed for fluconazole concentration using a high-performance liquid chromatography-UV method. PK parameters representing the extent (AUC0−∞) and rate (CmaX and Tmax) of absorption of fluconazole were obtained. An analysis of variance, a power analysis, 90% CI, and two 1-sided tests were used for statistical analysis of relative differences between the 2 drugs. Bioequivalence was concluded if the 90% CIs for the geometric mean ratios of AUC0−∞ and Cmax were between 0.80 and 1.25. A study investigator monitored the volunteers for adverse effects at 5 defined time points during the clinical part of the investigation.Thirteen men and 11 women (mean age, 1026 33.3 years; mean weight, 73.6 kg) completed the study. The respective point estimates of the ratios of geometric means of log-transformed Cmax and AUC00−∞ of fluconazole (test vs reference) were 0.985 and 1.047, with 90% CIs of 0.894 to 1.085 and 0.927 to 1.182, respectively. Differences in Tmax also did not reach statistical significance. No adverse effects were reported by the subjects or revealed by clinical or laboratory tests.The study failed to demonstrate any1026 statistically significant differences in Cmax and AUCO0−∞ values between the test and reference formulations of oral fluconazole 150 mg in this small, select population of healthy volunteers. On that basis, and according to both the rate and extent of absorption, the test and reference formulations were considered bioequivalent.

Rapid screening of toxic salbutamol, ractopamine, and clenbuterol in pork sample by high-performance liquid chromatography—UV method

A rapid and simple high-performance liquid chromatography–UV method was developed for the separation and quantification of salbutamol, ractopamine, and clenbuterol in pork. A mixture of acetonitrile–formic acid–ammonium acetate was used as the mobile phase to separate three β-agonists on a C18 column with gradient. The effects of the addition of formic acid and ammonium acetate to mobile phases on the separation of β-agonists were investigated. These additives can greatly improve the resolution and sensitivity. Under the optimized chromatographic condition, this separation does not need extra sample preparation. Complete baseline separation of three β-agonists was achieved in < 20 minutes; the linear range is 0.2–50 μg/L with a correlation coefficient R2 value of > 0.99. Excellent method reproducibility was found by intra- and interday precisions with a relative standard deviation of < 3%. The detection limit (S/N = 3) was found to be <0.05 μg/L; this method can be used for routine screening of the β-agonist residues in foods of animal origin before being identified by confirmatory methods.

Tetracycline Residues in Porcine Stomach after Administration via Drinking Water on a Swine Farm

Tetracycline is a broad-spectrum antibiotic used to treat infections in swine. The maximum residue levels of tetracycline in pork stomach tissue in Russia, Europe, and the United States are 10, 200, and 2,000 ppb, respectively. This difference in accepted safety levels may be the reason why stomach tissues that the United States exports continue to be residue violators in overseas markets. In this study, 30 pigs at two different stages of production (weanling and finisher) were treated with tetracycline at 22 mg/kg of body weight per day for a total of 5 days via a water medicator. Blood samples were collected at 0, 72, 78, 96, and 102 h after the start of medication. The medication was stopped at 120 h, and blood samples were again collected at 126, 144, 168, 192, and 216 h after exposure. Five animals were slaughtered for stomach tissue 0, 24, 48, 96, and 192 h after the drug was flushed from the water line. All blood and tissue samples were analyzed by high-performance liquid chromatography–UV methods. The tetracycline levels in plasma were below the level of detection after the U.S.-labeled withdrawal time of 4 days. The stomach tissue residues averaged 671.72, 330.31, 297.77, 136.36, and 268.08 ppb on withdrawal days 0, 1, 2, 4, and 8, respectively. Using the U.S. Food and Drug Administration tolerance limit method and a population-based pharmacokinetic model with Monte Carlo simulation, a withdrawal interval was estimated. This study demonstrated that tetracycline residues are still detectable in the stomach tissues after the established United States withdrawal time of 4 days. These residue levels may explain why stomach tissues tested in Russia and Europe show positive residues for tetracycline, even though the meat may pass inspection here in the United States prior to export.

Development and validation of high performance liquid chromatography with a spectrophotometric detection method for the chemical purity and assay of nepafenac

The study is a proposition of the application of high performance liquid chromatography (HPLC) with a spectrophotometric UV range detector to analyze the chemical purity and assay of nepafenac, an active pharmaceutical ingredient (API). During literature search only a few publications were found about nepafenac. HPLC UV methods were mainly presented in patent documents about nepafenac synthesis and chemical purity. The presented method allows to separate all potential related compounds from nepafenac and to quantitate the nepafenac amount. As there is no official monograph in the pharmacopeias about nepafenac, the performed full validation procedure makes the method ready to use in routine analysis. The composition of the mobile phase (10mM ammonium formate, pH 4.1) and the HPLC column (Phenomenex Gemini-NX C18) were selected during the development step. Presented data confirm the benefits of the developed method. Four of the most potential impurities were validated as for the quantitative test and the rest of impurities were validated as for the limit test – according to ICH Q2(R1). The accuracy/recovery results for the chemical purity method are within 90–108%, in the case of assay studies from 99% to 101%; the limit of detection is as low as 15–30ng/mL. The linearity passes all statistical tests.

Simultaneous Quantification of Levetiracetam and Gabapentin in Plasma by Ultra-Pressure Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection

Gabapentin (Neurontin) and levetiracetam (Keppra) are anticonvulsants with novel structures and suggested therapeutic ranges of 2-10 mg/L and 6-20 mg/L, respectively. Gabapentin is also used extensively to manage neuropathic pain, and for this indication, wherein higher doses are prescribed, plasma concentrations of 15-30 mg/L are typical. Here, we describe a simple rapid assay to support therapeutic drug monitoring of gabapentin and levetiracetam in plasma by ultra-pressure liquid chromatography couples to tandem mass spectrometry (UPLC-MS/MS) detection. After the addition of internal standard and protein precipitation of patient plasma with methanol:acetonitrile in a 50:50 ratio, 1 μL of supernatant sample is injected onto an Acquity UPLC HSS T3, 1.8 μm, 2.1 × 50 mm (Waters) column. Elution occurs using a linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column is eluted into a Waters Acquity UPLC TQD, operating in a positive mode to detect gabapentin at transition 172.18 > 154.11, levetiracetam at 171.11 > 126, and internal standard (3-amino-2-naphthoic acid) at 188.06 > 170. Secondary transitions for each analyte are also monitored for gabapentin at 172.18 > 137.06, levetiracetam at 171.11 > 154, and internal standard at 188.06 > 115. Runtime is 1.5 minutes per injection with baseline resolved chromatographic separation. The analytical measurement ranges were 1-150 mg/L for gabapentin and for levetiracetam. Intra-assay imprecision by the coefficient of variance (CV) was less than 8% and interassay CV was less than 5% for both analytes, at 4 different concentrations. Results obtained from patient samples were compared with results generated by established high-performance liquid chromatography-UV methods with the following regression statistics: y = 1.12x - 0.77, r = 0.996, Sy, x = 0.89, and n = 29 for gabapentin and y = 0.991x + 0.70, r = 0.997, Sy, x = 2.24, and n = 30 for levetiracetam. No analytical interferences were identified. : In summary, a simple reliable UPLC-MS/MS method was developed and validated for routine clinical monitoring of gabapentin and levetiracetam.

Rapid LC-UV Analysis of Iohexol in Canine Plasma for Glomerular Filtration Rate Determination

A rapid high-performance liquid chromatography UV method and a simple sample preparation for analyzing iohexol in canine plasma, for evaluating glomerular filtration rate (GFR) and intestinal permeability, were developed and validated. Trifluoroacetic acid (TFA) was used for protein precipitation and iohexol extraction from plasma, followed by vortex mixing and centrifugation. As an internal standard, 4-aminobenzoic acid (para-aminobenzoic acid, PABA) was added. The supernatant (5 μL) was injected into a Zorbax SB-C18 LC column maintained at 50 °C. The mobile phase of the LC method was a water–methanol gradient at pH 3.0 adjusted with TFA. Fast LC measurement was achieved by using a rapid-resolution LC technique. Total run time was 13 min, and UV wavelength was set at 246 nm. Precision of the method was 0.2–9.0%, depending on the iohexol concentration in plasma. Recovery of iohexol from plasma was over 90%, and recovery of the internal standard 99.1 ± 1.4%. The calibration curve was linear (r = 0.9997) over iohexol concentrations of 2.5–150 μg mL−1 (n = 5). This method is fast, simple, reliable and applicable in clinical settings.

Development of a new SPME–HPLC–UV method for the analysis of nitro explosives on reverse phase amide column and application to analysis of aqueous samples

A rapid, simple, sensitive and accurate quantitative method has been developed for the determination of eleven nitroaromatic components by solid phase microextraction (SPME) coupled to high performance liquid chromatography (HPLC) with UV detection from aqueous samples. PDMS/DVB resin fiber (60 μm) was used for concurrent extraction of all the analytes from aqueous matrix. Static desorption was carried out in the desorption chamber of SPME–HPLC interface containing mobile phase; methanol:water 43:57 (v/v) with subsequent liquid chromatographic analysis at isocratic flow rate of 1.3 mL/min and detection at 254 nm. A reverse phase amide column (5 μm) was used as a separation medium. The limit of detection (S/N = 3) for TNT and Tetryl was found to be 0.35 and 0.54 ng/mL, respectively. Developed method has been applied successfully to the analysis of aqueous samples obtained from environmental and industrial sources like river water, ground water, drinking water and industrial waste water.

Enantioselective and Nonlinear Intestinal Absorption of Eflornithine in the Rat

This study aimed to investigate if the absorption of the human African trypanosomiasis agent eflornithine was stereospecific and dose dependent after oral administration. Male Sprague-Dawley rats were administered single doses of racemic eflornithine hydrochloride as an oral solution (750, 1,500, 2,000, or 3,000 mg/kg of body weight) or intravenously (375 or 1,000 mg/kg of body weight). Sparse blood samples were obtained for determination of eflornithine enantiomers by liquid chromatography with evaporative light-scattering detection (lower limit of quantification [LLOQ], 83 microM for 300 microl plasma). The full plasma concentration-time profile of racemic eflornithine following frequent sampling was determined for another group of rats, using a high-performance liquid chromatography-UV method (LLOQ, 5 microM for 50 microl plasma). Pharmacokinetic data were analyzed in NONMEM for the combined racemic and enantiomeric concentrations. Upon intravenous administration, the plasma concentration-time profile of eflornithine was biphasic, with marginal differences in enantiomer kinetics (mean clearances of 14.5 and 12.6 ml/min/kg for L- and D-eflornithine, respectively). The complex absorption kinetics were modeled with a number of transit compartments to account for delayed absorption, transferring the drug into an absorption compartment from which the rate of influx was saturable. The mean bioavailabilities for L- and D-eflornithine were 41% and 62%, respectively, in the dose range of 750 to 2,000 mg/kg of body weight, with suggested increases to 47% and 83%, respectively, after a dose of 3,000 mg/kg of body weight. Eflornithine exhibited enantioselective absorption, with the more potent L-isomer being less favored, a finding which may help to explain why clinical attempts to develop an oral treatment have hitherto failed. The mechanistic explanation for the stereoselective absorption remains unclear.

A simple method for quantification of allopurinol and oxipurinol in human serum by high-performance liquid chromatography with UV-detection

Objectives Allopurinol is a uric acid lowering drug used in the treatment of gout and the prevention of tumor lysis syndrome. Allopurinol and its active metabolite oxipurinol inhibit xanthine oxidase, which forms uric acid from xanthine and hypoxanthine. Therapeutic drug monitoring is an important option for evaluation and optimization of allopurinol treatment in case of renal impairment, interaction with uricosuric drugs or to verify patient adherence. In this study we developed and validated a simple quantitative assay using reverse phased high-performance liquid chromatography (HPLC) with UV-detection as a method for quantification of allopurinol and oxipurinol in human serum in the presence of different frequently used drugs. Methods The HPLC–UV method uses a mobile phase consisting of sodium acetate (0.02 M; pH 4.5), at a flow rate of 1.0 mL/min. Allopurinol and oxipurinol are detected by UV-absorption at 254 nm with a retention time of 9.9 min for oxipurinol and 12.3 min for allopurinol. Aciclovir is used as internal standard. Results Validation showed for allopurinol lower and upper limits of quantification of 0.5 and 10 mg/L and for oxipurinol 1 and 40 mg/L, respectively. The assay was linear over the concentration range of 0.5–10 mg/L (allopurinol) and 1–40 mg/L (oxipurinol). Intra- and inter-day precision showed coefficients of variation <15% over the complete concentration range; accuracy was within 5% for allopurinol and oxipurinol. Endogenous purine-like compounds were separated from allopurinol, oxipurinol and aciclovir with a resolution factor >1.5. Exogenous purine-like compounds and co-medication frequently used by gout patients did not hinder the analysis due to the dichloromethane washing step or to low UV-absorpion at 253 nm. Serum levels of 66 patients prescribed allopurinol 300 mg/day were determined using this HPLC–UV method. Measured serum allopurinol and oxipurinol concentrations in clinical practice showed large variability with a range of <0.5–4.3 mg/L for allopurinol and <1.0–39.2 mg/L for oxipurinol, respectively. Conclusion We developed an easy-to-operate and validated HPLC–UV method for the quantification of allopurinol and oxipurinol in human serum. This method was proven to be valid for samples of gout patients frequently using concomitant medications.

HPLC Determination of DCJW in Rat Plasma and Its Application to Pharmacokinetics Studies

A high-performance liquid chromatography–UV method for determining DCJW concentration in rat plasma was developed. The method described was applied to a pharmacokinetics study of intramuscular injection in rats. The plasma samples were deproteinized with acetonitrile in a one-step extraction. The HPLC assay was carried out using a VP-ODS column and the mobile phase consisting of acetonitrile–water (80:20, v/v) was used at a flow rate of 1.0 mL min−1 for the effective eluting DCJW. The detection of the analyte peak area was achieved by setting a UV detector at 314 nm with no interfering plasma peak. The method was fully validated with the following validation parameters: linearity range 0.06–10 μg mL−1 (r > 0.999); absolute recoveries of DCJW were 97.44–103.46% from rat plasma; limit of quantification, 0.06 μg mL−1 and limit of detection, 0.02 μg mL−1. The method was further used to determine the concentration–time profiles of DCJW in the rat plasma following intramuscular injection of DCJW solution at a dose of 1.2 mg kg−1. Maximum plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC) for DCJW were 140.20 ng mL−1 and 2405.28 ng h mL−1.