High-performance Liquid Chromatography-ultraviolet Method Research Articles

Simple High-Performance Liquid Chromatography–Ultraviolet Method To Quantify the Molecular Size Distribution of Nonylphenol Ethoxylates

Polyethoxylated nonylphenols (NPEOx) are widely used in various domestic and industrial applications. These commercial products are mixtures of oligomers that affect aquatic ecosystems, acting as endocrine disruptors. In the present work a simple high-performance liquid chromatography–ultraviolet method to quantify single oligomers of nonylphenol ethoxylates was developed. The following NPEOx commercial mixtures were analyzed: Igepal CO-520, Igepal CO-630, and Igepal CO-720. Obtained results demonstrate that the absorbance of each oligomer was not a function of the ethoxyl chain length. This result allowed the comparison between experimental molecular distribution functions of NPEOx mixtures with molecular distributions predicted by the Poisson’s equation. In all cases, good agreement was obtained. The method was validated by quantifying NPEOx samples with different molecular size distributions.

Development and Validation of a High-Performance Liquid Chromatography Ultraviolet Method for Lapatinib Quantification in Human Plasma

A selective and precise high-performance liquid chromatography ultraviolet method was developed and validated for the determination of lapatinib in human plasma. After protein precipitation with acetonitrile, lapatinib and sorafenib were separated using isocratic elution (on a C18 Ultrabase column using a mobile phase of acetonitrile/20 mM ammonium acetate in a proportion 53:47 (v/v) pumped at a constant flow rate of 1.2 mL/min). Quantification was performed at 260 nm. Calibration curves were linear over the range 0.2-10 µg/mL. Inter- and intraday coefficients of variation were less than 7%. The limit of detection and the lower limit of quantification were 0.1 and 0.2 µg/mL, respectively. Recoveries of lapatinib from plasma were higher than 86.7% in all cases. The assay was applied to the determination of the drug in the plasma of 2 cancer patients receiving lapatinib, 1000 and 1250 mg orally, and could be useful for therapeutic drug monitoring of lapatinib in routine clinical practice.

RP-HPLC-UV Determination of ezetimibe in serum: Method development, validation and application to patients chronically receiving the drug

Summary Ezetimibe is the first in a new class of antihypercholesterolemic drugs. Since it has not long been available on the market, many of its properties may still be revealed. Analytical methods for its determination are scarce, especially regarding serum samples. A simple, fast, and effective high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the determination of ezetimibe concentration in human serum has therefore been developed. Three mobile phases were analysed, and original modifications to the concentration and flow parameters were made. Of five potential internal standards (IS), only nitrendipine was found to be suitable. The analytical wavelength was chosen based on the absorption spectrum of ezetimibe in the mobile phase. Finally, an extraction analysis was performed using two different solvents, and the extrahent volume was optimised. The final method developed was as follows. Single extraction of 1 mL serum sample, spiked with IS, was performed using 10 mL of methyl-t-butyl ether. Separation was obtained at ambient temperature on a Waters C18 Symmetry Shield (4.6 mm × 250 mm, 5 μm) column. The isocratic mobile phase was composed of acetonitrile and 0.1 M ammonium acetate aqueous solution 55:45 (v/v), set at flow rate of 0.75 mL min−1. Ezetimibe was detected at a wavelength of 232 nm after 5.49 min, and the IS was detected at 8.05 min. The developed method has been validated according to ICH standards. It was found to be specific, precise, accurate and linear over the range 10–800 ng mL−1 with R2 > 0.998, and detection and quantification limits of 4.60 ng mL−1 and 13.94 ng mL−1, respectively. The method has been applied to clinical serum samples. The developed technique allowed for successful in vivo assessment of ezetimibe concentrations in samples obtained from hypercholesterolemia patients who are chronically receiving the drug.

Ion-pair RP-HPLC method for the estimation of eberconazole nitrate in bulk form and in pharmaceutical dosage forms

Summary An isocratic ion-pair reversed phase high-performance liquid chromatography-ultraviolet (RP-HPLC-UV) method for analysis of eberconazole nitrate in bulk and in pharmaceutical dosage forms has been developed and validated. Best separation was achieved on Lichrospher C18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase of 10 mM potassium dihydrogen phosphate containing 10 mM tetra-butyl ammonium hydroxide (pH adjusted to 2.8 with ortho phosphoric acid) and methanol (75:25, v/v) at a flow rate of 1.0 mL min−1. UV detection was performed at 220 nm. The method was validated for specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness, and solution stability. The calibration plot was linear over the concentration range of 10–80 μg mL−1 (r2 = 0.999) and the limits of detection and quantification were 0.3 and 0.9 μg mL−1, respectively. Intra-day and inter-day precisions were 1.13% and 1.67%, respectively. Experimental design was employed to optimize the metho...

Determination of telaprevir in plasma of HCV‐infected patients by HPLC‐UV

Telaprevir is a direct acting antiviral agent, used with pegylated interferon and ribavirin for the management of chronic hepatitis C virus (HCV) genotype 1 infection, in patients not responding to therapy with pegylated interferon and ribavirin only. Although 75% of patients achieve a sustained virological response after treatment with telaprevir, adverse drug-drug interactions and undesirable events often occur. Therefore, telaprevir monitoring is pivotal to improve the management of HCV infection. Here, the first High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method to quantify telaprevir in human plasma of HCV-genotype 1-infected patients is reported. The volume of the plasma sample was 700 μL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone). The extracted samples were reconstituted with 150 μL of 60/40 water/acetonitrile. Thirty microliters of these samples was injected into a HPLC-UV system, and the analytes were eluted on a X Terra(®) RP18 column (250 mm × 4.6 mm i.d.) with a particle size of 5 μm. The mobile phase (ammonium acetate buffer, 150 mM, pH 8.0, and methanol:acetonitrile 50:50) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 16 min; telaprevir was detected by UV at 276 and 286 nm. The calibration curve was linear from 312.5 to 20,000 ng/mL (r(2) > 0.996). The absolute recovery of telaprevir ranged between 89 and 93% at concentrations of quality control samples of 800, 4,000, 8,000, and 16,000 ng/mL. Both precision and accuracy were always <15%. The HPLC-UV method reported here: (i) has been validated, (ii) is currently applied to monitor telaprevir in plasma of HCV-infected patients, and (iii) appears useful in a routine laboratory. ,

English

The main purpose of this study was to predict the efficacy, potency and sensitivity of meloxicam (a preferential cyclooxygenase-2 (COX-2) inhibitor) antipyretic effect by using a simple indirect response model in rat, evaluated by Brewer&rsquo;s yeast induced model. The rats received 1, 3, 7 and 10 mg/kg of meloxicam, after&nbsp;subcutaneous&nbsp;(sc) injection of Brewer&rsquo;s yeast. The plasma concentrations of meloxicam were determined by high performance liquid chromatography-ultraviolet (HPLC-UV) method. Rectal temperature (Ta) was measured for the assessment of the pharmacodynamic (PD) of the meloxicam. Before injection of yeast, basal fever mediator&rsquo;s synthesis (prostaglandin E2;&nbsp;PGE2) is maintained by physiological mechanism to regulate body temperature which is described by a constant rate synthesis (Ksyn) and a first order degradation of Kout. Ksyn&nbsp;is calculated by the equation, Ksyn&nbsp;= E0&nbsp;Kout, where E0&nbsp;is the baseline body temperature. After injection of yeast, the additional fever mediators&rsquo; synthesis is regulated by input rate (IR (t)). This process is governed by a first order rate constant (KIN), which can be inhibited by meloxicam. The pharmacokinetic (PK) parameters showed dose proportionality, with a Vd (4124.52, 4236.73, 4657.15, and 5912.1 ml/kg), CL (78.55, 149.25, 1313.57, and 1519.41 ml/h/kg), and Cmax (84.72, 258.29, 547.74, and 617.85 ng/ml). Indirect response PD model (inhibitory Emax&nbsp;model), estimated KIN&nbsp;(1.43, 0.63, 0.51, and 0.42 1/h), Kout&nbsp;(0.005, 0.008, 0.015, and 0.028 1/h), and Ksyn&nbsp;(0.29, 0.42, 0.076, and 0.03 h); estimates for IC50&nbsp;(concentration of meloxicam in plasma eliciting half of maximum inhibition of IR(t) or KIN) were 146.19, 379.51, 645.05, and 676.44 ng/ml of 1, 3, 7 and 10 mg/kg dose received by groups, respectively. This model appropriately describes the time course of pharmacological response to meloxicam to various doses, in terms of its mechanism of action and pharmacokinetics. &nbsp; Key words:&nbsp;Brewer&rsquo;s yeast, cyclo-oxygenase-2, fever mediators (PGE2), meloxicam, pharmacokinetic/ pharmacodynamic modeling.

In vitro–in vivo correlation study for the dermatopharmacokinetics of terbinafine hydrochloride topical cream

Background: To investigate the relationship between dermatopharmacokinetic (DPK) tape stripping from in vitro and in vivo using 1% terbinafine hydrochloride topical cream as the model formulation.Methodology: In vitro and in vivo tape strippings were conducted on separated pig ear skin used as a biological membrane for franz diffusion cell testing and the non-hairy skin area at the ventral forearms of healthy volunteers, respectively. Terbinafine (1%) topical cream was applied to the skin for 0.5, 2, and 4 h. The drug profiles of terbinafine across the stratum corneum were determined immediately (time 0 h), and at 0.5, 1, 2, and 4 h after removing the formulation. The amounts of terbinafine were analyzed by a validated high-performance liquid chromatography-ultraviolet method. The area under the curve (AUC) and the maximum amounts of terbinafine absorption (Qmax) were obtained from pharmacokinetic software. Partition coefficient (KSC/veh) and diffusion parameter (D/L2) were derived from the Fick’s second law equation. During the schedule time of 8 h, the deviations of in vitro and in vivo data were 6.61 and 30.46% for AUC and Qmax, respectively. There was insignificant difference of the KSC/veh and the D/L2 between excised pig ear and human skin. In addition, KSC/veh and D/L2 at Tmax of 2 h were used to predict the AUC presented the value of 4.7481 %h whereas the true value calculated from pharmacokinetic software provided the value of 5.9311 %h differing from each other in approximate of 20%.Conclusions: In vitro tape stripping using the separated pig ear skin as a viable membrane of the franz diffusion cell testing demonstrates the potential to represent in vivo tape stripping in human for topical bioavailability/bioequivalence study of terbinafine hydrochloride 1% topical cream.

Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Process Related Impurities and Degradation Products of Rasagiline Mesylate in Pharmaceutical Formulation

A sensitive, stability-indicating gradient reverse phase high-performance liquid chromatography-ultraviolet method has been developed for the quantitative determination of process-related impurities and forced degradation products of rasagiline mesylate in pharmaceutical formulation. Efficient chromatographic separation was achieved on an ACE C8, 150 × 4.6 mm, 3 µm column with mobile phase containing a gradient mixture of solvents A and B. The flow rate of the mobile phase was 0.8 mL/min with column temperature of 30°C and detection wavelength at 210 nm. Rasagiline was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Rasagiline was found to degrade significantly in acid and thermal stress conditions. The degradation products were well resolved from rasagiline and its impurities. The peak purity test results confirmed that the rasagiline peak was homogenous and pure in all stress samples and the mass balance was found to be more than 97%, thus proving the stability-indicating power of the method. The developed method was validated according to the guidelines of the International Conference on Harmonization with respect to specificity, linearity, limits of detection and quantification, accuracy, precision and robustness.

Determination of protopine in rat brain after oral administration of the extract of Corydalis decumbentis

A simple and sensitive high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed to determine protopine in rat brain samples after oral administration of the extract of Corydalis decumbentis. The extract of C. decumbentis was administered at a dose of 6.88 g/kg (equivalent to 159.62 mg/kg of protopine). The lower limit of quantification was 18.9 μg/ml for protopine. The calibration curve was linear over a concentration range of 21.6 to 324 μg/ml. Brain tissues samples were obtained at regular time intervals after oral administration of C. decumbentis extract. Protopine could be detected rapidly in rat brain after 30 min of oral administration and reach maximum concentration at about 10 h in male rat and 6 h in female rat. Furthermore, there was a great difference between male and female rats. In female rats, the maximum concentration was about twice that of male rats. This results obtained illustrate the pharmacokinetic behavior of protopine in brain tissue, and maybe helpful to explain the C. decumbentis bioactivity in cerebrovascular disease.

Chiral separation of cathinone derivatives used as recreational drugs by HPLC‐UV using a CHIRALPAK® AS‐H column as stationary phase

Cathinone derivatives gained high popularity on the recreational drugs market during the past 10 years. All these compounds are chiral, and the pharmacological potency of the enantiomers of these stimulants is supposed to differ. The goal of this research was to develop a reliable and easy-to-perform high-performance liquid chromatography ultraviolet method for the chiral separation of a set of 24 cathinone derivatives. A commercially available CHIRALPAK® AS-H column consisting of amylose tris [(S)-α-methylbenzylcarbamate] coated on 5-µm silica gel was found to be suitable to resolve a majority of the tested compounds. High-performance liquid chromatography measurements were performed in normal phase mode under isocratic conditions with a mobile phase consisting of hexane, isopropanol, and triethylamine at a flowrate of 1 ml/min. The ratio between hexane and isopropanol was optimized by means of three model substances. Under final conditions with a mobile phase of hexane, isopropanol, and triethylamine (97:3:0.1), 19 out of 24 compounds were successfully resolved into their enantiomers and detected at a wavelength of 254 nm. A correlation between the substituents of the nitrogen atom and the separation results are shown. Furthermore, enantiomer separation results of four cathinone derivatives were compared with the results of their amphetamine analogs.

Determination of CMPO using HPLC–UV

Octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO) is an extractant proposed for selective separation of radionuclide metals from used nuclear fuel solutions using solvent extraction. Radiolysis reactions can degrade CMPO and reduce separation performance and hence methods for measuring the concentration of CMPO are needed. A novel high performance liquid chromatography (HPLC) method was developed for measuring CMPO in dodecane that featured a low pH buffer, octanol as a co-solvent with 2-propanol, and ultraviolet (UV) detection. Validation data indicated that the HPLC–UV method for CMPO determination provided good linearity, sensitivity, accuracy and precision. Method performance was evaluated using CMPO samples that had undergone radiolysis, and the results showed a decrease in CMPO concentration and the appearance of degradation products. The degradation products were identified using electrospray ionization mass spectrometry, which also showed formation of CMPO–nitric acid complexes that account for the apparent loss of CMPO in an acidic environment, independent of irradiation.

Pharmacokinetic drug interaction between gemfibrozil and sitagliptin in healthy Indian male volunteers

To study the impact of gemfibrozil co-administration on the pharmacokinetics of sitagliptin in healthy Indian male volunteers. A randomized open label two-period crossover study involving 12 healthy Indian male volunteers was conducted at a single center. In each phase, the volunteers were administered sitagliptin as 100 mg tablets, either alone or co-administered with gemfibrozil as 600 mg tablets twice daily for 3 days. There was a 2-week washout period between phases. The venous blood samples were serially collected at 0-12 h post-dose, and plasma concentrations of the study drugs were estimated by a validated high-performance liquid chromatography-ultraviolet method. Relative to the administration of sitagliptin alone, co-administration with gemfibrozil increased the AUC₀₋₁₂ (2,167 ± 82.9 vs. 2,970 ± 76.4 ng h/ml; p < 0.0001), AUC(0-∞) (3,621 ± 222.5 vs. 5,574 ± 249.6 ng h/ml; p < 0.0002), C(max) (282.9 ± 7.7 vs. 344.1 ± 5.9 ng/ml; p < 0.0001), and t(½) (7.4 ± 0.6 vs. 10 ± 0.6 h; p = 0.0076) to statistically significant levels. The interindividual differences in the pharmacokinetic parameters of sitagliptin were found to be within acceptable limits (coefficient of variation <20%). No adverse drug events associated with sitagliptin occurred in the subjects during the study period. Although the bioavailability of sitagliptin was increased by 54% when co-administered with gemfibrozil, this interaction may not have any clinical significance as sitagliptin has a wide therapeutic index. Hence, in clinical practice, sitagliptin as 100 mg tablets and gemfibrozil as 600 mg tablets may be co-prescribed without much threat of sitagliptin toxicity. However, these results may not hold if the dose of sitagliptin is increased or if is co-prescribed with other antidiabetic drugs and/or cytochrome P450 2C8/human organic anion transporter-3 inhibitors. Further studies are needed to confirm these results in patients.

Development and validation of simple isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) method for determination of safflower yellow in Carthamus tinctorius (L.)-loaded nanostructured lipid carriers (NLC)

Carthamus tinctorius L. (safflower) is the traditional plant that is widely used in many regions around the Asia, including Thailand. With many reported biological activities, our group decided to formulate the cosmeceutical containing the florets extract of the plant. For enhancing the bioavailability of absorption of substances from its florets, the special carrier; nanostructured lipid carriers or nanostructured lipid carriers (NLC) was used. The simple isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) method for determination of safflower yellow, which is the main constituent in the florets, was developed and validated to determine the potency of the formulated products. Cold methanol precipitation technique was used to extract unentrapped safflower from the wax matrix of NLC. For HPLC analysis, 60% methanol was used as a mobile phase with 1 ml/min flow rate, detected by UV detector at 401 nm. Accuracy and precision of the developed method was determined over the concentration range of 5 to 100 ppm. Specificity of the method was confirmed by high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS). Linear range of the analysis was found in the concentration ranged of 5 to 100 ppm safflower yellow. Lower limit of detection and quantitation were 1.4 and 4.1 ppm, respectively. The developed method will be useful in the pharmaceutical and cosmeceutical industry applications concerning NLC containing safflower extract. Key words: Cosmeceutical, high performance liquid chromatography-ultraviolet (HPLC-UV), nanostructured lipid carriers (NLC), safflower, safflower yellow.

Simultaneous determination of valproic acid and 2-propyl-4-pentenoic acid for the prediction of clinical adverse effects in Chinese patients with epilepsy

Valproic acid (VPA) is a well-established anticonvulsant drug that has been increasingly used in the treatment of many forms of generalized epilepsy. Although there are many reports of adverse effects of VPA, studies focusing on the concentration–response relationships of VPA and its metabolites in patients with epilepsy are extremely limited. In this study, a rapid and specific high performance liquid chromatography-ultraviolet (HPLC-UV) method to simultaneously detect the concentrations of VPA and its major hepatotoxic metabolite 2-propyl-4-pentenoic acid (4-ene VPA) in human plasma has been established, using 2,4′-dibromoacetophenone and octanoic acid as the derivatization reagent and internal standard, respectively. This method was used to analyze plasma samples (n=64) of Chinese patients with epilepsy. The results revealed that 4-ene VPA concentrations in Chinese patients were much higher than those in patients in other countries such as United States and Iran. Significant correlations between aspartate aminotransferase (AST), alanine aminotransferase (ALT) and 4-ene VPA concentration suggest that the simultaneous determination of VPA and 4-ene VPA is an effective tool for the prediction of clinical hepatotoxicity in epileptic patients. Furthermore, the present study describes a less costly and complex technique for the clinical monitoring of VPA plasma levels and the risk of hepatotoxicity which may be of particular interest in developing countries like China.

English

19F NMR provided a highly specific tool for identification of efavirenz in human serum and pharmaceutical preparations as well as a suitable analytical technique for their absolute quantification. The method is based on the integration of appropriate signals of efavirenz and potassium fluoride, as an internal standard. The proposed method is simple and reliable, but when it is used without any sample pretreatment, there is a manipulation of large sample volumes and lengthy analysis time. The calibration curve for efavirenz in human serum was linear over the range of 0.8 to 1000 mgL&minus;1, with detection limit of 0.3ï€ mgL&minus;1. The mean error on human serum samples ranged from about -4 to -6%, with relative standard deviations of &lt;8%. The method was also applied successfully for the determination of efavirenz in real pharmaceutical samples,and compared with the results obtained by a reference high-performance liquid chromatography-ultraviolet (HPLC-UV) method. &nbsp; Key words:&nbsp;Efavirenz,&nbsp;19F NMR analyses, HIV, serum, pharmaceuticals.

Analysis of CoQ10 in Cultivated Tobacco by a High-Performance Liquid Chromatography–Ultraviolet Method

Coenzyme Q (CoQ) is a naturally occurring lipid-soluble quinone that performs multiple functions in all living cells and has become a popular antioxidant supplement, a coadjuvant in the treatment of heart disease, and the object of study for treating neurodegenerative disorders. Although there are many tools for CoQ analysis of microbial and animal samples, there have been relatively few reports of methods for CoQ analysis of green plants. This work describes a method for the routine analysis of coenzyme Q(10) in green leaf tissue of cultivated Nicotiana tabacum (tobacco) using high-performance liquid chromatography (HPLC) with UV detection. The method was applied to the analysis of CoQ(10) in N. tabacum 'KY14' leaves at different stalk positions representing young lanceolate to senescing leaves, and it was found that CoQ(10) increased as leaf position changed down the stalk from 18.69 to 82.68 μg/g fw. The method was also used to observe CoQ(10) in N. tabacum 'NC55' and N. tabacum 'TN90LC' leaves over time, finding that CoQ(10) leaf content remained relatively stable from 3 to 6 weeks but increased in both cultivars at 8 weeks. This method will likely be useful in the analysis of CoQ(10) in the green leaves of other plant species.

Residue Depletion of Nitrovin in Chicken after Oral Administration

In this study, the residue depletion of nitrovin in chicken was studied after feeding the birds with dietary feeds containing 10 mg/kg of nitrovin for 7 consecutive days. Tissues (muscle, fat, kidney, and liver) and plasma were collected at different withdrawal periods and determined by a high-performance liquid chromatography-ultraviolet (HPLC-UV) method. The limit of detection for nitrovin in tissue and plasma samples was 0.1 ng/(g or mL), and the inter- and intrarecoveries from the blank fortified samples were in the range of 71.1-85.7%. At the withdrawal period of 0 days, the residue concentration of nitrovin in plasma was the highest (average of 84.98 ng/mL) compared to those in muscle, fat, liver, and kidney (average of 21.04, 61.18, 24.04, and 68.28 ng/g, respectively). At the withdrawal period of 28 days, the residue levels of nitrovin in muscle, fat, liver, and plasma were all higher than 1.0 ng/(g or mL) and the highest concentration was in liver (average of 5.8 ng/g). These data are in support of the ban of nitrovin as a feed additive in food-producing animals.

PARAFAC based methods for the analysis of Diltiazem drug excitation emission matrices of fluorescence obtained by a derivatization reaction

A fluorescent derivatization reaction for Diltiazem drug quantification based on the condensation reaction of citric or malonic acid with acetic anhydride, catalyzed by the tertiary amine group of Diltiazem, was developed. Excitation emission matrices (EEMs) of fluorescence of the pure solvent (ethanol), standard and sample solutions following a standard addition methodology were analysed by PARAFAC based methods (PARAFAC, PARAFAC2 and PARALIND) to obtain robust calibration methodologies. The quantification results of the sample were compared with the official US Pharmacopeia high performance liquid chromatography-ultraviolet method (USP HPLC-UV). Although the experimental sets of EEM show linearity deviations all the PARAFAC based methods allow correct robust estimation of Diltiazem concentration in pharmaceutical formulations. The closest results were: derivatization with citric acid and PARAFAC2 six components non-negativity constraint model with a detection limit of 0.088 ppm; and, derivatization with malonic acid and PARAFAC six components non-negativity constraint model with a detection limit of 0.066 ppm for the malonic acid was observed. The simultaneous utilization of the three PARAFAC methods gives further information about the intrinsic structure of the data sets under analysis, i.e., it works as an efficient diagnostic tool for the existence of non-linearity and colinearity.

A Validated High-Performance Liquid Chromatography-Ultraviolet Method for Quantification of the CCR5 Inhibitor Maraviroc in Plasma of HIV-Infected Patients

Maraviroc is the first commercialized CCR5 inhibitor for HIV therapy. A new high-performance liquid chromatography-ultraviolet method to quantify maraviroc concentrations in human plasma was developed and validated. The method is based on a protein precipitation procedure, with an acidic solution of acetonitrile (trifluoroacetic acid 0.1%) and quinoxaline as internal standard. The analytes were eluted using a gradient run in 15 minutes on an analytical C18 Luna column (150 mm x 4.6 mm ID) with a particle size of 5 mum. Maraviroc and internal standard were detected by UV at 193 nm and 352 nm, respectively. The calibration curve was linear up to 2500 ng/mL. The mean recovery of maraviroc was 96%. All validation data were in accordance with U.S. Food and Drug Administration requirements. The new high-performance liquid chromatography-ultraviolet method reported here could be used routinely to monitor plasma concentrations of maraviroc in healthy volunteers and HIV-infected patients.

Investigation of the Crossreactivity of Mycophenolic Acid Glucuronide Metabolites and of Mycophenolate Mofetil in the Cedia MPA Assay

The immunosuppressant mycophenolic acid (MPA) used for solid organ transplantation is predominantly metabolized to a pharmacologically inactive phenolic glucuronide (MPAG) and, to a lesser extent, to the pharmacologically active acyl glucuronide (AcMPAG). The recently introduced CEDIA Mycophenolic Acid Assay from Microgenics has been reported to overestimate MPA in clinical samples and crossreactivity with AcMPAG has been suspected. A detailed investigation of the crossreactivity of AcMPAG and the prodrug mycophenolate mofetil (MMF) in the CEDIA assay is presented using pure substances. In addition, MPA concentrations in plasma were compared with a validated high-performance liquid chromatography-ultraviolet method. Plasma samples from kidney (KTx, n = 50), heart (HTx, n = 50), and liver (LTx, n = 50) transplant recipients were analyzed by the CEDIA (MPA) and a high-performance liquid chromatography-ultraviolet method (MMF, MPA, MPAG, AcMPAG). Crossreactivity of MMF (0.93-46.3 mg/L), MPAG (50-1000 mg/L), and AcMPAG (0.5-10 mg/L) was investigated using spiked drug-free plasma. Method comparison was performed using Bland & Altman and Passing & Bablok analysis. The method bias was correlated to AcMPAG concentrations using Spearman's rank correlation. Crossreactivity with AcMPAG and MMF was concentration-dependent and reached 215% and 143%, respectively. There was no crossreactivity with MPAG. The CEDIA assay showed a mean positive bias of 36.3% in patient samples. The mean bias was lowest with HTx samples (15%), 41.7% with KTx samples, and highest with LTx samples (52.3%). There was a positive correlation between the method bias and AcMPAG concentrations (r = 0.829; P < 0.001). No MMF was detected in patient samples. The CEDIA overestimates MPA concentrations on average by 36%. This bias is mainly the result of AcMPAG as previously observed with the EMIT MPA assay. It should be considered that the putative therapeutic range for MPA with the CEDIA assay will be higher than the range using high-performance liquid chromatography.