High-pressure Liquid Chromatographic Assay Research Articles

Pharmacokinetic Characterization of Intravenous Cyclosporine Treatment for Cardioprotection During Resuscitation of Asphyxiated Newborn Piglets

Cyclosporine treatment, as a single intravenous bolus, during resuscitation has been shown to attenuate myocardial injury in asphyxiated newborn piglets. However, the pharmacokinetics of cyclosporine treatment for cardioprotection in newborns has not been studied. We aimed to assess the pharmacokinetics of a single intravenous cyclosporine treatment during resuscitation of asphyxiated newborn piglets and compare these parameters with healthy newborn piglets. Newborn piglets were acutely instrumented and normocapnic alveolar hypoxia was induced for 2 hours followed by 4 hours of reoxygenation. Piglets were block-randomized to receive a single intravenous bolus of cyclosporine (2.5-25 mg/kg) (n = 8 per group). Eight piglets underwent no hypoxia-reoxygenation and received 10 mg/kg cyclosporine at the corresponding time point. Plasma cyclosporine and troponin concentrations during reoxygenation period were determined by high-pressure liquid chromatography and enzyme-linked immunosorbent assay, respectively. Noncompartmental methods were used to calculate the pharmacokinetic parameters. Cyclosporine concentrations and pharmacokinetic parameters were analyzed by one-way analysis of variance. University animal laboratory. Piglets (1-4 days old, weighing 1.4-2.5 kg). Intravenous cyclosporine (2.5, 10, or 25 mg/kg) given during resuscitation. In the hypoxic-reoxygenated piglets, the plasma AUC(0-4 hrs) and C(max) of cyclosporine at reoxygenation were in the following rank order: 25 > 10 > 2.5 mg/kg treatment (p < 0.001 between groups, analysis of variance). Plasma AUC(0-4 hrs) and C(max) in piglets treated with cyclosporine at 25 mg/kg was associated with increased plasma troponin levels, a marker of myocardial injury, relative to piglets treated with 2.5 and 10 mg/kg. Asphyxiated newborn piglets had higher clearance and lower AUC(0-∞), but similar AUC(0-4 hrs), steady-state volume of distribution, and mean residence time compared with those of healthy newborn piglets. This is the first study to demonstrate the pharmacokinetics of intravenous cyclosporine treatment during resuscitation of asphyxiated newborn piglets, which did not appear to different from that of healthy piglets.

Pharmacokinetics of Meropenem in Critically Ill Patients With Severe Infections

Meropenem is an effective β-lactam antibiotic that is frequently used to treat serious infections in both intensive care unit (ICU) and febrile neutropenic hematology/oncology (Hem/Onc) patients. Studies suggest that to be effective, meropenem concentrations must be maintained above the inhibitory concentrations for the majority of a dosing interval. However, the pharmacokinetics (PK) of meropenem seem to differ in critically ill patients compared with healthy or less ill subjects used to select labeled dosing regimens. This study was designed to investigate meropenem PK in critically ill patients and to see how often standard dosing regimens produced adequate plasma concentrations. A secondary objective was to investigate how achieved concentrations were related to outcomes (morbidity and mortality) in these patients. Meropenem plasma concentrations over time were measured using a high pressure liquid chromatography assay in febrile Hem/Onc and ICU patients who were treated with standard meropenem dosing schedules. Outcomes such as fever control and survival were assessed in these patients and compared with individual meropenem PK data and with recommended target concentrations. A total of 25 subjects including 10 febrile Hem/Onc and 15 ICU patients with a variety of serious illnesses and baseline renal function were studied. Mean peak concentrations were less variable than were pre-dose concentrations. Post peak and trough concentrations were often below recommended minimum inhibitory concentrations. Both clearance and volumes of distribution were greater than reported in less ill subjects, only in part explained by increased renal clearance. Therefore, serum concentrations often did not exceed recommended concentration targets even for moderately sensitive organisms. Inadequate concentrations were especially common in the mostly ill, febrile neutropenic Hem/Onc subjects and seemed to explain at least some therapeutic failures. Conversely, drug accumulation occurred in ICU subjects with decreased renal function. Standard meropenem dosing regimens were inadequate in many critically ill febrile, neutropenic Hem/Onc, and septic ICU patients. These data suggest a role for meropenem concentration monitoring in such patients.

Monitoring trough voriconazole plasma concentrations in haematological patients: real life multicentre experience

The objective of this retrospective study was to evaluate results from voriconazole therapeutic drug monitoring (TDM) in haematological patients in routine clinical practice. Between 2005 and 2010, 1228 blood samples were obtained from 264 haematological patients (median 3 samples/patient; range 1-27) receiving voriconazole for targeted/preemptive treatment of invasive aspergillosis (IA) (46.3% of samples), empirical therapy (12.9%) or prophylaxis (40.8%). A high-pressure liquid chromatography assay was used to analyse voriconazole concentrations. Clinical and laboratory data were analysed retrospectively. The median of the detected voriconazole plasma concentration was 1.00 μg ml(-1) (range <0.20-13.47 μg ml(-1)). Significant inter- and intra-patients variability of measured concentrations (81.9% and 50.5%) were identified. With the exception of omeprazole administration, there was no relevant relationship between measured voriconazole concentrations and drug dose, route administration, age, gender, CYP2C19*2 genotype, gastrointestinal tract abnormality, administration via nasogastric tube, serum creatinine, and liver enzymes. However, per patient analysis identified significant role of individual voriconazole dose and drug form change on measured plasma concentration. Measured voriconazole concentrations did not correlate with the treatment outcome of patients with IA. We only identified a limited number of adverse events related to voriconazole therapy; however, the median plasma concentration was not different from concentrations measured in samples without reported toxicity. Our retrospective study has suggested that routine monitoring of voriconazole plasma concentrations has probably only a limited role in daily haematological practice.

Novel biodegradable sandwich-structured nanofibrous drug-eluting membranes for repair of infected wounds: an in vitro and in vivo study

BackgroundThe purpose of this study was to develop novel sandwich-structured nanofibrous membranes to provide sustained-release delivery of vancomycin, gentamicin, and lidocaine for repair of infected wounds.MethodsTo prepare the biodegradable membranes, poly(D, L)-lactide-co-glycolide (PLGA), collagen, and various pharmaceuticals, including vancomycin, gentamicin, and lidocaine, were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol. They were electrospun into sandwich-structured membranes with PLGA/collagen as the surface layers and PLGA/drugs as the core. An elution method and a high-pressure liquid chromatography assay were used to characterize in vivo and in vitro drug release from the membranes. In addition, repair of infected wounds in rats was studied. Histological examination of epithelialization and granulation at the wound site was also performed.ResultsThe biodegradable nanofibrous membranes released large amounts of vancomycin and gentamicin (well above the minimum inhibition concentration) and lidocaine in vivo for more than 3 weeks. A bacterial inhibition test was carried out to determine the relative activity of the antibiotics released. The bioactivity ranged from 40% to 100%. The nanofibrous membranes were functionally active in treating infected wounds, and were very effective as accelerators in early-stage wound healing.ConclusionUsing the electrospinning technique, we will be able to manufacture biodegradable, biomimetic, nanofibrous, extracellular membranes for long-term delivery of various drugs.

English

The acetylation polymorphism is one of the most common inherited variations in the biotransformation of drugs and chemicals and large number of studies has been done to determine the distribution of acetylator phenotypes among populations of different geographic origin. The aim of this study was to investigate the acetylator phenotypes of the Pakistani population and compare it between male and female healthy volunteers. The polymorphic acetylation of sulphamethazine has been investigated in male and female volunteers (50 each) of Pakistani population. Sulphamethazine was administered orally in Capsule form as 500 mg to each healthy male and female volunteer. Sulphamethazine and acetylsulphamethazine were determined in the six hour plasma samples by&nbsp;high-pressure liquid chromatography (HPLC). Acetylator phenotype was determined from the metabolic ratio of acetylsulphamethazine to sulphamethazine in the plasma samples. The acetylation of sulphamethazine by&nbsp;arylamine N-acetyltransferase 2 (NAT2) showed bimodal population frequency distribution. 60% of the female volunteers were found to be fast and 40% to be slow acetylators while that of male volunteers were 62% fast and 38% slow acetylators. &nbsp; Key words:&nbsp;NAT2 acetylation phenotype, sulphamethazine, male &amp; female volunteers, HPLC assay.

Oxidative mechanisms for the biotransformation of 1-methyl-1,6-dihydropyridine-2-carbaldoxime to pralidoxime chloride

AimsDue to pralidoxime chloride's (2-PAM) positive charge, it's penetration through the blood brain barrier (BBB) and reactivation of organophosphate (OP) inhibited central nervous system (CNS) acetylcholinesterase (AChE) is poor. The results of CNS inhibited AChE are seizures. Pro-2-PAM (1-methyl-1,6-dihydropyridine-2-carbaldoxime), a pro-drug of 2-PAM, due to higher hydrophobicity, penetrates the BBB better but must be oxidized to 2-PAM, the active form of the oxime to reactivate CNS AChE in order to abrogate seizures. In this study, we characterize the in vivo mechanism of pro-2-PAM oxidation. Main methodsA high pressure liquid chromatography (HPLC) assay was developed to quantify the conversion of pro-2-PAM to 2-PAM. NADPH oxidase activity was measured by a photo-luminescence assay using lucigenin substrate. Upon analysis, the rate of NADPH induced oxidation suggested that an alternate mechanism may be involved. Therefore, various enzyme co-factors of oxidation–reduction enzyme systems were evaluated, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), riboflavin 5′-phosphate (FMN), and riboflavin. Next, a spectrophotometric assay was developed to measure the conversion of pro-2-PAM to 2-PAM in the presence of riboflavin. Key findingsIn guinea pig brain homogenate, diphenyleneiodonium (DPI), a specific NADPH oxidase inhibitor, reduced pro-2-PAM to 2-PAM conversion to less than 25%. In contrast, riboflavin, FAD, and FMN rapidly oxidized all pro-2-PAM to 2-PAM in an in vitro assay. Riboflavin oxidized pro-2-PAM reactivated diisopropylfluorophosphate (DFP) inhibited AChE. SignificanceThe present study shows that pro-2-PAM was rapidly oxidized by riboflavin to 2-PAM, which reactivated organophosphate (OP)-inhibited AChE.

Kidney function and retinol status in type 2 diabetes mellitus patients

Kidneys play an important role in retinol turnover. We postulated that retinol homeostasis is disturbed in diabetic nephropathy. The aim of this research was to study the effect of kidney impairment on urinary excretion and on serum concentrations of retinol in type 2 diabetes mellitus patients. For this purpose, 41 type 2 diabetes patients and 9 sex -and age-matched healthy subjects were enrolled. Serum and urinary retinol and retinol-binding protein (RBP) were assessed by high-pressure liquid chromatography and enzyme-linked immunosorbent assay, respectively. The study showed that 17 out of 41 diabetic patients (41.5%) and none of the controls excreted retinol in urine (P<0.02). Retinol excretion in the urine in these patients was 1.5-fold more prevalent than hypercreatininemia. Urinary retinol significantly correlated with clinically diagnosed nephropathy (P=0.02). All but one of the patients with hypercreatininemia excreted retinol in the urine. Serum retinol and RBP in patients with hypercreatininemia were higher than in controls (P<0.002). Values of urinary retinol, unlike urinary RBP, albumin and total protein, did not overlap between patients and controls. Our results indicate that (i) urinary retinol is a specific sign of tubular damage in type 2 diabetic patients and (ii) urinary retinol enables a more clear-cut identification of proximal tubule dysfunction in type 2 diabetes patients than urinary RBP or albumin.

Elution Characteristics of Doxorubicin-loaded Microspheres Differ by Drug-loading Method and Microsphere Size

Purpose To examine the loading and elution behavior of doxorubicin and superabsorbent polymer microspheres (SAP-MS) as they relate to particle size and loading techniques. Materials and Methods SAP-MS, 30–60 μm and 50–100 μm, were subject to loading 50 mg of doxorubicin from a dry lyophilized state. Doxorubicin loading was performed after prehydration of SAP-MS (one-step method) or serially in two divided administrations (two-step method). Loading rate and elution characteristics were determined after doxorubicin analysis using a high-pressure liquid chromatography (HPLC) assay. All experiments were performed in triplicate. Results All systems showed the ability to load and elute doxorubicin effectively in the specified time frame (loading 15 minutes to 2 hours and elution 1 hour to 14 days). For the two loading methods, 30–60 μm SAP-MS showed no statistically significant difference in loading rate but a statistically significant difference in cumulative elution at 14 days (19.13 mg vs 17.83 mg, one-step vs two-step; P = .02). For the two loading methods, 50–100 μm SAP-MS showed no statistically significant difference in loading rate and no statistically significant difference in cumulative elution at 14 days (14.87 mg vs 12.77 mg, one-step vs two-step; P = .20). Conclusions SAP-MS exhibit the ability to load and release doxorubicin. In comparing particle size and loading methods, higher cumulative elution rates were associated with smaller (30–60 μm) particle size and one-step loading. Higher elution from the one-step loading method may be due to release of unbound doxorubicin. Differences in the loading and elution of doxorubicin may depend on the increased surface area of smaller SAP-MS resulting in alterations of behavior of doxorubicin and its interactions with the polymer microspheres.

Cree antidiabetic plant extracts display mechanism-based inactivation of CYP3A4

Seventeen Cree antidiabetic medicinal plants were studied to determine their potential to inhibit cytochrome P450 3A4 (CYP3A4) through mechanism-based inactivation (MBI). The ethanolic extracts of the medicinal plants were studied for their inhibition of CYP3A4 using the substrates testosterone and dibenzylfluorescein (DBF) in high pressure liquid chromatography (HPLC) and microtiter fluorometric assays, respectively. Using testosterone as a substrate, extracts of Alnus incana, Sarracenia purpurea, and Lycopodium clavatum were identified as potent CYP3A4 MBIs, while those from Abies balsamea, Picea mariana, Pinus banksiana, Rhododendron tomentosum, Kalmia angustifolia, and Picea glauca were identified as less potent inactivators. Not unexpectedly, the other substrate, DBF, showed a different profile of inhibition. Only A. balsamea was identified as a CYP3A4 MBI using DBF. Abies balsamea displayed both NADPH- and time-dependence of CYP3A4 inhibition using both substrates. Overall, several of the medicinal plants may markedly deplete CYP3A4 through MBI and, consequently, decrease the metabolism of CYP3A4 substrates including numerous medications used by diabetics.

The Comparison between Two Methods for Typing of Nontuberculous Mycobacteria: High Pressure Liquid Chromatography and Molecular Assay Genotype Mycobacterium CM/AS

The GenoType Mycobacterium CM and the GenoType Mycobacterium AS (HAIN Lifescience, Germany) were evaluated for the ability to differentiate mycobacterial species of clinical isolates. Serial use of the both assays is aimed to identify 38 different molecular patterns, of which 24 patterns can be assigned to single species, 10 patterns are allocated to two or more Mycobacterium species, and 4 patterns correspond to Mycobacterium species and gram-positive bacteria with a high G + C content. The analysis of mycolic acids by high pressure liquid chromatography (HPLC) was the reference method. A set of 127 nontuberculous mycobacterial isolates on Loewenstein-Jensen slants, derived from different patients between 1999 and 2007, was analyzed. The strains were primary classified by HPLC following the diagnostic procedure, and retyped by GenoType Mycobacterium CM/AS. In total, results obtained by both methods were interpretable for 113 strains. Concordant results were obtained for 105 (93%) mycobacterial strains. One out of 8 inconcordant classified strains, which was classified as M. abscessus/M. chelonae by HPLC, displayed a pattern of M. tuberculosis complex by a molecular method. Eleven clinical strains were differentiated only by one of used methods, either by HPLC (6 strains) or by GenoType CM/ AS (5 strains). Three strains were not classified at all. Our results show that the GenoType Mycobacterium CM/AS system represents a useful tool to identify mycobacterial clinical isolates. The molecular system is as rapid and reliable as the HPLC, but much easier to perform and more friendly for the environment.

Disposition of morphine in tissues of the pregnant rat and foetus following single and continuous intraperitoneal administration to the mother.

Foetal exposure to maternally administered opiates such as morphine represent a serious human health problem but disposition studies in man are difficult to perform. Morphine disposition was therefore investigated in pregnant rats and their foetuses near term as a model. Disposition was examined either following intraperitoneal dosing as a single dose or continuous infusion. A high-pressure liquid chromatography assay for morphine in plasma and tissue was developed and validated. Following the single morphine dose, foetal distribution was rapid and concentrations in foetal and placental tissue were from 2.6 (whole foetus) to 27.6 (placenta) times higher compared with maternal plasma. The rank order of the area under the concentration vs time curve (AUC) of morphine in tissues was: placenta > or = foetal liver > foetal brain > whole foetus > maternal brain. The foetal brain to maternal brain AUC ratio for morphine was 9.5, suggesting large differences in their blood-brain barrier permeability. Following continuous administration of morphine there were significant linear relationships between maternal plasma and tissue concentrations with the same rank order as the single dose study. However, following continuous administration the relative amount of morphine in placenta and foetal liver was reduced by half and one-third, respectively, compared with the single dose study. These results document why the rat foetus is particularly susceptible to the pharmacodynamic effects of morphine following maternal administration.

Ultra high-pressure liquid chromatographic assay of moxifloxacin in rabbit aqueous humor after topical instillation of moxifloxacin nanoparticles

The present report describes a rapid and sensitive ultra high-pressure liquid chromatography (UHPLC) method with UV detection to quantify moxifloxacin in rabbit aqueous humor. After deproteinisation with acetonitrile, gradient separation of moxifloxacin was achieved on a Waters Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm) column at 50 °C. The mobile phase consisted of 0.1% trifluoroacetic acid in water and acetonitrile at a flow rate of 0.4 ml/min. Detection of moxifloxacin was done at 296 nm. Method was found to be selective, linear ( r = 0.9997), accurate (recovery, 97.30–99.99%) and precise (RSD, ≤1.72%) in the selected concentration range of 10–1000 ng/ml. Detection and quantitation limit of moxifloxacin in aqueous humor were 0.75 and 2.5 ng/ml, respectively. The aqueous humor levels of moxifloxacin after single topical instillation in three formulations, i.e. moxifloxacin solution (Moxi-SOL), anionic nanoparticles (Moxi-ANP) and cationic nanoparticles (Moxi-CNP) were investigated. A fourfold increase in the relative bioavailability was observed with the Moxi-CNP (AUC 0→ t , 3.14 μg h/ml) compared with Moxi-SOL (AUC 0→ t , 0.79 μg h/ml) and Moxi-ANP (AUC 0→ t , 0.72 μg h/ml) formulation. The results indicate that the cationic nanoparticle increases ocular bioavailability of moxifloxacin and prolong its residence time in the eye.

Immunodosage de l'hémoglobine glyquée sur ci8200 (Abbott Diagnostics) en présence d'hémoglobines anormales

The objective of this study was to compare haemoglobin A(1c) (HbA(1c)) levels measured by the immunoturbidimetric assay on ci8200 Architect (Abbott Diagnostics) to those obtained by the high pressure liquid chromatography (HPLC) assay from Tosoh Bioscience. Firstly, we verified correlation between the two methods in 47 subjects without hemoglobin variants: the coefficient of correlation was 0.968 and the regression equation was as follows: y(Abbott) = 0.928x(Tosoh) - 0.081. We then measured HbA(1c) levels by both methods in blood samples of 23 patients with a structural hemoglobin variant (A/S, A/C or S/C). Analysis of these samples revealed significant discrepancies between results of both methods, as indicated by the higher mean value obtained by immunoturbidimetric assay when compared to HPLC assay (7.17 +/- 2.68% vs. 5.86 +/- 1.41%). Classification of patients according to the HAS (French Haute Autorité de Santé) recommendations HbA(1c) was not modified for 68% of patients with abnormal hemoglobin. However, in 32% of cases, discrepancies between the two methods may have clinical importance, which justifies the occurrence of an abnormal hemoglobin when the HbA(1c) assay is performed by the method immunoturbidimetry Abbott Diagnostics.

Extracorporeal Photophoresis Augments Function of CD4+CD25+FoxP3+ Regulatory T Cells by Triggering Adenosine Production

During extracorporeal photophoresis (ECP), peripheral blood mononuclear cells are treated with DNA-intercalating agents and irradiated with ultraviolet light. This procedure exerts immunosuppressive effects, most likely mediated by regulatory T cells (Treg). However, the underlying mechanisms are not clear yet. In our study, we investigated the effect of ECP on frequency and function of Treg in the peripheral blood of patients suffering from graft-versus-host disease. Whole blood samples from graft-versus-host disease patients were taken before and after the ECP treatment on 2 consecutive days. Phenotypical analysis of changes in distinct leukocyte subsets within the peripheral blood of patients and healthy controls was performed by means of flow cytometry. Functional analysis of the Treg population after magnetic bead isolation was performed using conventional suppression assays, and adenosine was detected by means of high pressure liquid chromatography and Lanzetta assays. We show that the frequency of CD4/CD25/FoxP3 Treg in the peripheral blood increases after each cycle of ECP and also in the course of treatment. The suppressive capacity of Treg after ECP was increased compared with that of Treg before ECP, although not reaching the suppression levels obtained with Treg from healthy controls. Furthermore, we show that ECP stimulates the CD39-mediated production of adenosine by Treg, which substantially reduces the T-cell proliferation in in vitro suppression assays. Our data indicate that ECP stimulates the conversion of ATP to adenosine by the ectonucleotiodase CD39, which acts as a novel soluble immunosuppressive reagent mediating immunosuppression of Treg.

Effects of St. John's wort extract on indinavir pharmacokinetics in rats: Differentiation of intestinal and hepatic impacts

Aims To evaluate the possible herb–drug interaction of St. John's wort (SJW) extracts with indinavir in rats and to set up a model for characterizing pre-systemic sites for the interactions between orally administered herbs and pharmaceuticals. Main Methods The in vivo pharmacokinetic study and in situ single-pass intestinal perfusion model were employed in the research. Plasma indinavir concentration and cytochrome P 450 3A activities were measured by high-pressure liquid chromatography and spectrophotometric assays, respectively. Key findings Oral administration of either 150 or 300 mg/day SJW for 15 days significantly reduced indinavir plasma levels with certain pharmacokinetic parameter changes. The cytochrome P 450 3A analysis suggested that this interaction was attributable to the induction of indinavir metabolism. Further perfusion study demonstrated that both small intestine and the liver contributed significantly to the reduction of indinavir bioavailability and was flow rate-dependent. Moreover, the small intestine was the major site for the pre-systemic metabolism of indinavir, whether with or without SJW pretreatment. Significance Herb–drug pharmacokinetic interactions between SJW and indinavir can be clearly observed in the Wistar rat model. Particularly, the respective first-pass effect contributed by the small intestine and the liver could be differentiated and quantified. The application of the animal model to investigating herb–drug interactions or other relevant research purposes is envisioned.

Enantiomeric disposition of ketorolac in goats following administration of a single intravenous and oral dose

The purpose of this study was to investigate the stereospecific pharmacokinetics of ketorolac (KT) in goats following a single 2 mg/kg intravenous (i.v.) dose and a single 6 mg/kg oral dose. A stereoselective high pressure liquid chromatography assay was used to quantify ketorolac plasma concentrations. Pharmacokinetic parameters for both stereoisomers were estimated by model independent methods. Following an i.v. dose, the plasma concentration profiles for the stereoisomers were similar with half-lives of 1.05 +/- 0.62 h for R-KT and 1.05 +/- 0.61 h for S-KT. Clearance values for R- and S-KT after an i.v. dose were 0.53 +/- 0.23 and 0.54 +/- 0.23 L.h/kg, respectively. Following an oral dose, the terminal half-lives were longer with values of 34.08 +/- 11.81 and 33.97 +/- 12.19 h for R-KT and S-KT, respectively. The average bioavailability was 133 +/- 23% for R-KT and S-KT, respectively. The longer half-lives and high apparent bioavailability after oral dosing are suggestive of a slow absorption process in the gastrointestinal tract and recycling. The results indicate that interconversion of the stereoisomers of ketorolac is absent in goats. However, studies with individual isomers are needed before any conclusion can be drawn about the lack of bioinversion.

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19 x − 1.4 for tazobactam and y = 1.7 x − 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.

Measurement of Plasma Concentration of Gemcitabine and Its Metabolite dFdU in Hemodialysis Patients with Advanced Urothelial Cancer

We investigated the pharmacokinetics of gemcitabine and its metabolite in two male patients (52 and 56-year-old) with advanced urothelial cancer receiving hemodialysis three times a week. Gemcitabine, 1000 mg/m(2) in 100 ml of saline, was intravenously administered for 30 min. The concentration of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) was measured at several given time points using a high-pressure liquid chromatography assay. Pharmacokinetic parameters were determined using the two-compartment modeling program. Gemcitabine was rapidly eliminated from plasma even in patients with renal dysfunction. No obvious differences in pharmacokinetic parameters such as the t(1/2), AUC and C(max) of gemcitabine were observed between the patients on hemodialysis and those with normal renal function in previous reports. On the other hand, dFdU showed a sustained level until hemodialysis was initiated. Hemodialysis could reduce the plasma dFdU level by approximately 50%. According to the previous information, no dose modification of gemcitabine may be required for patients with renal impairment or hemodialysis. However, gemcitabine should be given with caution because only limited information is available, and the clinical effect of sustained and/or accumulated dFdU is unknown.

Comparison of routine test kits of creatinine in human serum with reversed-phase high-pressure liquid chromatographic assay

目的 观察人血清肌酐反相HPLC测定的不精密度和准确性,以HPLC为标准,评价几种人血清肌酐常规测定方法的准确性.方法 Beckman LX20测定系统采用Jaffe动力学法,强生Vitros 250测定系统采用干化学法,酶法采用协和酶法试剂盒,在Hitatchi7170全自动生化分析仪上进行测定人血清肌酐.肌酐HPLC测定以反相C18为固定相,5-氟尿嘧啶(5-Fluorouracil,5-Fu)为内标,于235 nm处进行检测.结果 HPLC法的批内不精密度(n=5)＜2.0%,日间不精密度(n=20)＜5.1%.绝对回收率＞95.31%,相对回收率＞98.95%.不同肌酐浓度有证参考物质的测定值与靶值一致,平均偏倚为-0.31%～1.35%.HPLC法作为独立变量,当肌酐浓度＜200 μmol/L时,Beckman LX20系统、强生干化学系统及酶法的偏倚分别为-19%～21%、-14%～21%和-30%～11%;当肌酐浓度＞200 μmol/L时,Beckman LX20系统、强生干化学系统及酶法的偏倚分别为-10%～13%、-13%～14%和-20%～10%,其中酶法结果基本为负偏倚,在肌酐浓度70 μmol/L时,有的负偏倚达到30%.结论 以5-Fu为内标的人血清肌酐反相高压液相色谱法精密度和准确性好.Beckman LX20和强生测定系统与反相高压液相色谱法的偏倚相对较小,酶法测定结果系统偏低,且当血清肌酐浓度＜70 μmol/L时不能准确测定。

Determination of microsomal CYP2A6 activity by high performance liquid chromatography

Coumarin has been reported to be a reliable probe for the determination of human microsomal CYP2A6 activity. Coumarin is converted to 7-hydroxycoumarin by CYP2A6. A high pressure liquid chromatographic assay for the estimation of CYP2A6 activity in microsomes was evaluated. A RP C-18 Novapak Waters (15 cm x 3.9 mm, 5 µm) column was used for the assay. The mobile phase composition was methanol : 1% glacial acetic acid (35:65 v/v) (pH~3.1), with a flow rate of 0.6 ml/min, injection volume of 100 µl and detection at 320 nm. The retention times for coumarin and 7-hydroxycoumarin were 8.7 min and 5.3 min, respectively. The limit of detection (LOD) was 0.05 µM, and the limit of quantitation (LOQ) was 0.1 µM, for 7-hydroxycoumarin. The percent coefficient of variation associated with 7-hydroxycoumarin determination after duplicate estimation was found to be in the range 0.03 to 3.6% for buffer matrix and 0.1 to 6.5% for microsomal matrix. The mean rate of 7-hydroxycoumarin formation in guinea pig liver microsomes was 0.084 nmol/min/nmol P450. Coumarin 7-hydroxylase activity was absent in rat liver microsomes. No interference was observed from incubation mixture components.