Journal of Chromatography B (v.835, #1-2)
Editorial Board (iii).
High-performance liquid chromatographic determination of the magnetic resonance imaging contrast agent Gadocoletate ion in human plasma, urine and faeces by Ilaria Setti; Roberto Celeste; Giancarlo Mariani; Daniela Dal Fiume; Alberto Morisetti; Vito Lorusso (1-15).
Gadocoletate ion is a new paramagnetic intravascular contrast agent for magnetic resonance imaging (MRI). An high-performance liquid chromatographic method for assaying Gadocoletate ion in human plasma, urine and faecal samples is described. The analysis is based on the reversed-phase chromatographic separation of Gadocoletate ion from the endogenous components of the biological matrices and its detection during elution by ultraviolet light absorption at 200 nm. The selectivity of the method was satisfactory. The mean absolute recovery during the analytical sample preparation was greater than 87%. The precision, expressed as coefficient of variation (CV%) ranged from 0.29 to 5.90% and the accuracy, expressed as mean relative error (R.E.%) of the analytical method ranged from −3.7 to +7.1%. The detection limit in plasma and urine was 2.01 and 10.0 μg/mL (0.00203 and 0.0101 μmol/mL), respectively. The detection limit in homogenized faecal samples was 17.7 μg/g (0.0179 μmol/g). Stability studies were performed in human plasma and urine samples during the analytical cycle. Gadocoletate ion was shown to be stable in human plasma and in human urine when stored at about +4 °C for up 24 h, and after three freeze-thaw cycles. In addition, it was shown to be stable in samples of processed plasma and in diluted urine at about +4 °C for 48 h, and at room temperature for at least 24 h. As regards the long-term stability of Gadocoletate ion, the results of dedicated studies showed that Gadocoletate ion is stable in human plasma samples when stored at +4 °C for up to 30 days and at −80 °C for up to 90 days. Gadocoletate ion is stable in samples of human urine when stored at +4 °C for up to 30 days, and when stored at −20 °C and at −80 °C for up to 90 days. The method has been successfully validated in human plasma, urine and faeces and it has been shown to be precise, accurate and reliable.
Keywords: Gadocoletate trisodium; MRI; HPLC analysis; Gadolinium complex; ICP–AES;
Development and validation of isomer specific RP-HPLC method for quantification of cefpodoxime proxetil by Vasu Kumar Kakumanu; Vinod Kumar Arora; Arvind Kumar Bansal (16-20).
The present work explains the development and validation of a simple and reliable isomer specific liquid chromatographic method for the quantitative determination of cefpodoxime proxetil (CP) in rat in situ intestinal perfusate samples. Chromatography was carried out by reversed-phase technique on a C-18 column with a mobile phase composed of 20 mM ammonium acetate buffer (pH 5.0) and acetonitrile in the ratio of 62:38 pumped at a flow-rate of 1 ml/min. The detection was carried out at 235 nm and a column temperature of 30 °C. The method was evaluated for the various validation parameters, such as linearity, accuracy, precision, LOD, LOQ, specificity, selectivity, and sample stability. The results of intra- and inter-day validation (n = 3) showed the method to be efficient and the same was applied in an in situ permeability study conducted for CP in rats.
Keywords: Racemic cefpodoxime proxetil; R, S-enantiomers; Separation of isomers; Reversed-phase; HPLC; Liquid chromatography;
Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography–mass spectrometry with electrospray ionization by Takashi Miyayama; Pei-Suen Tsou; Sun-Mi Fung; Ho-Leung Fung (21-26).
We have developed a liquid chromatographic–mass spectrometric method for the simultaneous determination of nitroglycerin (NTG) and its active metabolites, glyceryl 1,2-dinitrate (1,2-GDN) and glyceryl 1,3-dinitrate (1,3-GDN), for metabolism studies in cell cultures. 1,2,4-Butanetriol-1,4-dinitrate was chosen as an internal standard. Using a linear gradient of water/methanol containing 0.025 mM NH4Cl, the compounds were eluted within 12.5 min on an Allure Aqueous C18 column (100 mm × 2.1 mm). Detection and quantification was achieved with multiple reaction monitoring in the negative ion mode. Intra- and inter-day variabilities for simultaneous determination of the three nitrates were below 10 and 18%, respectively, over a range of NTG and GDN concentrations of 0.5–15 ng/ml. The lower limit of quantification was found to be about 0.01 ng on column. Application of this method was illustrated through in vitro metabolism studies of NTG in culture media bathing LLC-PK1 cells and human vascular smooth muscle cells (HA-VSMC) at 37 °C. The degradation half-life of NTG was found to be 4.5 ± 0.4 h and 39.2 ± 0.02 h, respectively, for LLC-PK1 cells versus HA-VSMC. At 5 h, the 1,2-GDN versus 1,3-GDN metabolite distribution ratio in the bathing medium was found to be 1.5 ± 0.1 and 0.2 ± 0.02 for LLC-PK1 and HA-VSMC cells, respectively. With this method, the degradation half-life of NTG in rat plasma at 37 °C was shown to be 26.8 ± 1.8 min, consistent with previous values obtained using gas chromatography.
Keywords: LC–MS; Nitroglycerin; Dinitrate; Metabolism; LLC-PK1 cells; Rat plasma; Human vascular smooth muscle cells;
Direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study by Hea-Young Cho; Chan-Ho Park; Yong-Bok Lee (27-34).
A simple, rapid, and accurate column-switching liquid chromatography method was developed and validated for direct and simultaneous analysis of loxoprofen and its metabolites (trans- and cis-alcohol metabolites) in human serum. After direct serum injection into the system, deproteinization and trace enrichment occurred on a Shim-pack MAYI-ODS pretreatment column (10 mm × 4.6 mm i.d.) by an eluent consisting of 20 mM phosphate buffer (pH 6.9)/acetonitrile (95/5, v/v) and 0.1% formic acid. The drug trapped by the pretreatment column was introduced to the Shim-pack VP-ODS analytical column (150 mm × 4.6 mm i.d.) using acetonitrile/water (45/55, v/v) containing 0.1% formic acid when the 6-port valve status was switched. Ketoprofen was used as the internal standard. The analysis was monitored on a UV detector at 225 nm. The chromatograms showed good resolution, sensitivity, and no interference by human serum. Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 15 and over 95%, respectively, in the concentration range of 0.1–20 μg/ml. With UV detection, the limit of quantitation was 0.1 μg/ml, and good linearity (r = 0.999) was observed for all the compounds with 50 μl serum samples. The mean absolute recoveries of loxoprofen, trans- and cis-alcohol for human serum were 89.6 ± 3.9, 93.5 ± 3.2, and 93.7 ± 4.3%, respectively. Stability studies showed that loxoprofen and its metabolites in human serum were stable during storage and the assay procedure. This analytical method showed excellent sensitivity with small sample volume (50 μl), good precision, accuracy, and speed (total analytical time 18 min), without any loss in chromatographic efficiency. This method was successfully applied to the pharmacokinetic study of loxoprofen in human volunteers following a single oral administration of loxoprofen sodium (60 mg, anhydrate) tablet.
Keywords: Loxoprofen; Alcohol metabolites; On-line column switching;
Quantitative determination of MCC-555, a novel insulin sensitizer in beagle dog plasma by high-performance liquid chromatography with fluorescence detection by Ning Sun; Mei Lin; Guorong Fan; Zhanying Hong; Guocai Lu (35-39).
A sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of MCC-555 (5-[[6-(2-fluorbenzyl)-oxy-2-naphy] methyl]-2,4-thiazolidinedione) in beagle dog plasma. Sample preparation was done by protein precipitation with acetonitrile and a synthetic intermediate of MCC-555 (5-[[6-(2-fluorbenzyl)-oxy-2-naphy] methylene]-2,4-thiazolidinedione) was used as the internal standard (IS). The isocratic mobile phase consisted of acetonitrile–10 mmol/l sodium phosphate buffer (pH 4.5) (65:35, v/v) was delivered at a flow rate of 1 ml/min to a Kromasil C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). The compounds were detected by fluorescence detection, using an excitation wavelength of 232 nm, and emission wavelength of 352 nm. Calibration curves of MCC-555 were linear in the concentration range of 0.005–2.0 μg/ml. Intra- and inter-day precision ranged from 3.4 to 5.4% and 3.0 to 8.8%, respectively. No endogenous interferences were observed with either MCC-555 or IS. The assay is simple, economical, precise, and is directly applicable to pharmacokinetic studies in beagle dogs involving three dose administrations.
Keywords: MCC-555; High-performance liquid chromatography; Pharmacokinetics;
Determination of the enantiomeric composition of a new insulin sensitizer in plasma samples from non-clinical and clinical investigations using chiral HPLC with electrospray tandem mass spectrometric detection by B. Lausecker; G. Fischer (40-46).
Two drug assays were developed and applied to assess the enantiomeric composition of an insulin sensitizer drug in plasma after administration of its racemate to man, and in human and animal plasma and serum samples generated after in vitro experiments. The sample preparation for the assays consisted either of protein precipitation and column-switching, or liquid–liquid extraction and direct injection. Subsequently, both assays employed chiral HPLC coupled to atmospheric pressure ionization mass spectrometry. An interconversion of the racemate to a mixture enriched with the (+)-enantiomer could be confirmed for all species and biological matrices. The individual enantiomers could be quantified in the concentration range 0.5–500 ng/ml, starting with a 100-μl plasma aliquot. Inter- and intra-assay precision and accuracy were in the range 0.1–7.9 and 88.8–106.0%, respectively. Run times of 5 min for a single sample allows the analysis of more than 200 samples overnight.
Keywords: Ionspray; Chiral drug assay; Glitazones; Interconversion;
Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human biomonitoring using isotope-dilution gas chromatography high-resolution mass spectrometry by Lovisa C. Romanoff; Zheng Li; Kisha J. Young; Nelson C. Blakely; Donald G. Patterson; Courtney D. Sandau (47-54).
In order to perform comprehensive epidemiological studies where multiple metabolites of several PAHs are measured and compared in low-dose urine samples, fast and robust methods are needed to measure many analytes in the same sample. We have modified a previous method used for measuring polycyclic aromatic hydrocarbon (PAH) metabolites by automating the solid-phase extraction (SPE) and including an additional eight metabolites. We also added seven new carbon-13 labeled standards, which improves the use of isotope-dilution calibration. Our method included enzyme hydrolysis, automated SPE and derivatization with a silylating reagent followed by gas chromatography (GC), coupled with high-resolution mass spectrometry (HRMS). Using this method, we measured 23 metabolites, representing 9 parent PAHs, with detection limits in the low pg/mL range. All steps in the clean-up procedure were optimized individually, resulting in a method that gives good recoveries (69–93%), reproducibility (coefficient of variation for two quality control pools ranged between 4.6 and 17.1%, N > 156), and the necessary specificity. We used the method to analyze nearly 3000 urine samples in the fifth National Health and Nutrition Examination Survey (NHANES 2001–2002).
Keywords: PAH; Polycyclic aromatic hydrocarbon; Biomonitoring; Urinary metabolite; Hydroxylated PAH; Method development;
Determination of monoamines in urine by capillary electrophoresis with field-amplified sample stacking and amperometric detection by Qianfeng Weng; Guowang Xu; Kailong Yuan; Ping Tang (55-61).
A simple, rapid and low-cost method using capillary electrophoresis coupled with field-amplified sample stacking and electrochemical detection was developed for the separation and determination of monoamines. In this present work, a systematic study of the parameters (pH value and concentration of electrophoretic buffer, composition of sample solvent, injection voltage and time) affecting separation and on-line concentration of monoamines has been performed enabling the detection sensitivity of these monoamines to be improved by 5000 times compared with the conventional electrokinetic injection. This developed method was applied to the direct analysis of these monoamines in human urine without off-line sample preconcentration. Due to the requirement for urine dilution to minimize the detrimental effects of high salt on analyte stacking, the real sensitivity improvement is about 50-fold when applying the optimized method to urine samples. In order to quantitate these monoamines accurately, internal standard calibration curves were constructed with standard monoamines in presence of salt with similar concentration as in human urine. In the method validation, the calibration curves were linear over a range of 1.0 × 10−9 to 2.5 × 10−8 mol/L for each monoamine and the limits of detection (signal to noise ratio of 3) for these monoamines were in the sub-nmol/L concentration range (6.0 × 10−10 mol/L).
Keywords: Capillary electrophoresis; Field-amplified sample injection; Amperometric detection; Monoamines; Urine;
Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex by Michael Beverly; Kim Hartsough; Lynn Machemer; Pam Pavco; Jennifer Lockridge (62-70).
The ocular metabolism of an siRNA duplex, SIRNA-027, was examined by ion-pair reversed-phase liquid chromatography (IP-RP-LC) coupled to electrospray ionization mass spectrometry (ESI–MS). The RNA duplex was injected intraocularly into the eyes of New Zealand white rabbits. Rabbits were sacrificed at different timepoints and the vitreous and retina/choroid tissue analyzed for siRNA by IP-RP-LC-MS. The method used a hexafluoroisopropanol (HFIP)/triethylamine (TEA) ion-pairing buffer with a methanol gradient. Using electrospray ionization, the duplex was preserved in the gas phase for analysis by a triple quadrupole mass spectrometer. With this methodology metabolites from rabbit ocular vitreous humor and retina/choroid tissue were identified and a pattern of siRNA degradation was established. Results showed that the duplex was metabolized predominantly from one end. This end of the siRNA duplex was calculated to have the weakest binding energy of the two ends indicating that the ability of the siRNA to split into single strands is a factor in its degradation.
Keywords: siRNA; HPLC; VEGF; Electrospray; RNAi; LC–MS; Metabolites; Oligonucleotides; Mass spectrometry; ESI;
Validated LC–MS–MS method for determination of m-nisoldipine polymorphs in rat plasma and its application to pharmacokinetic studies by Hairong Wang; Lantong Zhang; Qiao Wang; Zhifang Yuan; Min Li (71-76).
A sensitive and specific liquid chromatography–tandem mass spectrometric (LC–MS–MS) method has been developed to determine m-nisoldipine in rat plasma. Sample was pretreated by a single-step protein precipitation with acetonitrile, in contrast to the liquid–liquid procedure frequently used for the extraction of 1,4-dihydropyridines from biologic samples. Separation of analyte and internal standard (I.S.) was performed on a Symmetry RP-C18 analytic column (50 mm × 4.6 mm, 3.5 μm) with a mobile phase consisting of acetonitrile–water (80:20, v/v) at a flow rate of 0.5 ml/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) scan mode using TurboIonSpray ionization (ESI) source. The method was sensitive with a lower limit of quantification (LLOQ) of 0.2 ng/mL, with good linearity (r ≥ 0.9982) over the linear range of 0.2–20 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic and relative bioavailability studies of m-nisoldipine polymorphs in rats.
Keywords: m-Nisoldipine; Polymorphs; Liquid chromatography–tandem mass spectrometry;
Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli by Oscar Aguilar; Verónica Albiter; Leobardo Serrano-Carreón; Marco Rito-Palomares (77-83).
A direct comparison of a chromatography and an aqueous two-phase system (ATPS) processes for the partial purification of penicillin acylase (PA) produced by a recombinant strain of E. coli, was performed. An established chromatography process for the recovery of PA was selected as a model system and characterised for comparison with a developed ATPS prototype process. PEG-phosphate systems were selected for the recovery of PA over PEG-citrate systems, since higher enzyme recovery and increased purity was obtained. ATPS proved to be suitable to process highly concentrated disrupted extract (35%, w/w) and maintain a high top phase enzyme recovery. In the direct comparison of the processes, the superiority of the ATPS approach was highlighted since a reduction of the number of unit operations from 7 to 4 was achieved. An outline economic analysis based on the cost of separation agent of the processes favour the ATPS process, in which a gross cost reduction of 37% (from $0.47 to $0.30 USD) was achieved. Such result was obtained considering a potential re-use of up to 100 times of the resin used in the chromatography process. Additionally, by assuming that all the unit operations are equivalent in investment and operating cost, further reduction of approximately 43% of the respectively involved cost can be obtained when the ATPS process is used. Overall, the proposed ATPS process comprising of PEG1450-phosphate, tie-line length (TLL) of 48.5% (w/w), volume ratio (Vr) of 1.0, pH of 7.0 and 35% (w/w) PA crude extract loaded into the system proved to be more efficient, recovering 97% of PA at the top phase (PEG rich phase) with a purity factor of 3.5. It is clear that the results reported herein raise the consideration for the potential substitution of the chromatography process for PA recovery from E. coli as a first step for the development of an optimised and economic process with evident commercial application.
Keywords: Penicillin acylase recovery; Process comparison; Aqueous two-phase systems;
Simultaneous electrochemiluminescence determination of sulpiride and tiapride by capillary electrophoresis with cyclodextrin additives by Jianguo Li; Fengjuan Zhao; Huangxian Ju (84-89).
Sulpiride and tiapride are often used in the treatment of depression, schizophrenia and psychopathology of senescence, gastric or duodenal ulcers and are also partly excreted by kidney. This work developed a simple and sensitive method for their simultaneous monitoring in human urine based on capillary electrophoresis coupled with electrochemiluminescence detection by end-column mode. β-Cyclodextrin (β-CD) was used as an additive to the running buffer to obtain the absolute separation of sulpiride and tiapride. Under optimized conditions the proposed method displayed a linear range from 1.0 × 10−7 to 1.0 × 10−4 M for both sulpiride and tiapride with the correlation coefficients more than 0.995 (n = 6). Their limits of detection were 1.0 × 10−8 M (45 amol) and 1.5 × 10−8 M (68 amol) at a signal to noise ratio of 3, respectively. The relative standard deviations for six determinations of 2.0 μM sulpiride and 3.0 μM tiapride were 1.8 and 2.5%, respectively. For practical application an extract step with ethyl acetate at pH 11 was performed to eliminate the influence of ionic strength in sample. The recoveries of sulpiride and tiapride at different levels in human urine were between 84 and 95%, which showed that the method was valuable in clinical and biochemical laboratories for monitoring sulpiride and tiapride for various purposes.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Sulpiride; Tiapride; β-Cyclodextrin; Simultaneous determination;
A validated gas chromatographic–electron impact ionization mass spectrometric method for methylenedioxymethamphetamine (MDMA), methamphetamine and metabolites in oral fluid by Karl B. Scheidweiler; Marilyn A. Huestis (90-99).
An analytical method to simultaneously quantify amphetamine (AMP), methamphetamine (MAMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), 3-hydroxy-4-methoxy-methamphetamine (HMMA) and 3-hydroxy-4-methoxy-amphetamine (HMA) in oral fluid is presented. Four hundred microlitres of oral fluid collected via expectoration was extracted by solid phase extraction. GC/MS–EI with selected ion monitoring (SIM) yielded linear curves 5–250 ng/mL for AMP, MAMP, MDMA and MDEA, 5–500 ng/mL for MDA and 25–1000 ng/mL for HMA and HMMA. Recoveries were greater than 85%, accuracy 87–104%, and precision less than 8.3% coefficient of variation. This assay will be used to investigate distribution of sympathomimetic amines into human oral fluid following controlled drug administration.
Keywords: Amphetamine; Methamphetamine; Methylenedioxymethamphetamine; Ecstasy; Oral fluid; Gas chromatography; Mass spectrometry;
Simple and rapid high-performance liquid chromatographic method for determination of celecoxib in plasma using UV detection: Application in pharmacokinetic studies by A. Zarghi; A. Shafaati; S.M. Foroutan; A. Khoddam (100-104).
A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of celecoxib in human plasma. The analysis was carried out on a monolithic silica column using UV detection at 254 nm. The assay enables the measurement of celecoxib for therapeutic drug monitoring with a minimum quantification limit of 10 ng ml−1. The method involves simple, one-step extraction procedure, and analytical recovery was 100.5 ± 1.3%. The calibration curve was linear over the concentration range of 10–800 ng ml−1. The coefficients of variation for inter-day and intra-day assay were found to be less than 8%. We also demonstrate the applicability of this method for pharmacokinetic studies in humans.
Keywords: Celecoxib; Plasma; HPLC; Monolithic column;
Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography–mass spectrometry by Thomas F. Kalhorn; William N. Howald; Scott Cole; Brian Phillips; Joanne Wang; John T. Slattery; Jeannine S. McCune (105-113).
A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC–MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is ∼20 fmol. Plasma CEPM concentration is determined using LC–MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4 h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.
Keywords: Hydroxycyclophosphamide; Carboxyethylphosphoramide mustard; CEPM; LCMS; Chloride adduct; Phenylhydrazine; Plasma analysis; Drug targeting;
Determination of scutellarin in mouse plasma and different tissues by high-performance liquid chromatography by Fei Xiong; Hao Wang; Ji Cheng; Jiabi Zhu (114-118).
A simple HPLC–UV method was developed for the determination of scutellarin in plasma and different tissues of mice (heart, liver, spleen, lungs and kidneys). The separation was achieved by HPLC on a Hypersil C18 column with a mobile phase composed of methanol–water–glacial acetic acid (40:60:1). UV detection was used at 335 nm. The calibration curves were linear in all matrices (r 2 > 0.997) in the concentration range of 0.1–10 μg/ml for plasma and 0.1–20 μg/g for tissue homogenates, respectively. The method described is suitable for studies on the distribution of scutellarin in different tissues of mice.
Rapid determination of solanesol in tobacco by high-performance liquid chromatography with evaporative light scattering detection following microwave-assisted extraction by Hua-Ying Zhou; Chun-Zhao Liu (119-122).
Solanesol is the starting material for many high-value biochemicals, including coenzyme Q10 and Vitamin-K analogues. The aim of the current study was to develop a reliable and fast analytical procedure for the determination of solanesol in tobacco using high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique. The HPLC conditions were Agilent C18 column using acetonitrile–isopropanol (60:40, v/v) as mobile phase at a flow rate of 1 ml/min. ELSD conditions were optimized at nebulizer-gas flow rate of 1.5 l/min and drift tube temperature of 65 °C. The method was validated to achieve the satisfactory precision and recovery, and the calibration range was 0.1–1.5 mg/ml. The developed analytical procedure was successfully applied to determine solanesol content in tobacco samples from different growing regions in China.
Keywords: Evaporative light scattering detection; High performance liquid chromatography; Microwave-assisted extraction; Solanesol; Tobacco;
Sensitive high-performance liquid chromatographic quantitation of gabapentin in human serum using liquid–liquid extraction and pre-column derivatization with 9-fluorenylmethyl chloroformate by Gholamreza Bahrami; Amir Kiani (123-126).
Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum are based on the same approach, involving o-phthaldialdehyde derivatization of deproteinized serum samples. The present paper however, describes a new, simple and sensitive high-performance liquid chromatographic method for determination of gabapentin in human serum using liquid–liquid extraction and 9-fluorenylmethyl chloroformate (FMOC-Cl) as pre-column labeling agent. The drug and an internal standard (azithromycin) were extracted from serum by salting-out approach using a mixture of dichloromethane–2 propanol (1:1, v/v) as the extracting solvent. The extracted analytes were subjected to derivatization with FMOC-Cl in the presence of phosphate buffer (pH 7). A mobile phase consisting of methanol–0.05 M sodium phosphate buffer (73/27, v/v; pH of 3.9) containing 1 ml/l triethylamine was eluted and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm × 4.6 mm) column. The standard curve was linear over the range of 0.03–20 μg/ml and limit of quantification was 0.03 μg/ml. The performance of analysis was studied and the validated method showed excellent performance in terms of selectivity, specificity, sensitivity, precision and accuracy. No interferences were found from commonly co-administered antiepileptic agents.
Keywords: Reverse phase chromatography; HPLC; Gabapentin; Serum; 9-Fluorenylmethyl chloroformate; FMOC-Cl;
Validation of liquid/liquid extraction method coupled with HPLC-UV for measurement of ribavirin plasma levels in HCV-positive patients by Antonio D’Avolio; Allende Ibañez; Mauro Sciandra; Marco Siccardi; Daniel Gonzalez de Requena; Stefano Bonora; Giovanni Di Perri (127-130).
Measurement of ribavirin plasma levels in HCV-positive patients have been shown to be useful in order to optimise individual ribavirin exposure. Efficacy and toxicity of this drug are shown to be concentration-dependant. A simple HPLC-UV method was developed and validated, which has an easy liquid/liquid extraction, sensitive limit of detection, without any interference peaks, reproducible and linear over the range of clinical relevant concentrations. The assay warrants further evaluation as a tool for ribavirin therapeutic drug monitoring in HCV-positive patients.
Keywords: Ribavirin; Liquid/liquid; HPLC; UV method;
Simple and rapid liquid chromatography method for determination of efavirenz in plasma by Geetha Ramachandran; A.K. Hemanth Kumar; Soumya Swaminathan; P. Venkatesan; V. Kumaraswami; David J. Greenblatt (131-135).
A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C18 column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 μg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at −20 °C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.
Keywords: Efavirenz; Plasma; HPLC;
High-performance liquid chromatographic method for the determination of gemcitabine and 2′,2′-difluorodeoxyuridine in plasma and tissue culture media by Mark N. Kirstein; Iman Hassan; Dan E. Guire; Dennis R. Weller; Jason W. Dagit; James E. Fisher; Rory P. Remmel (136-142).
Gemcitabine, a pyrimidine antimetabolite undergoes metabolism by plasma and liver cytidine deaminase to form the inactive compound, 2′,2′-difluorodeoxyuridine (dFdU). The parent molecule is activated by intracellular phosphorylation. To evaluate the population pharmacokinetics in patients receiving gemcitabine, and to test the relation between gemcitabine infusion rate and antitumor activity in an in vitro bioreactor cell culture system, we developed and validated a sensitive and specific HPLC-UV method for gemcitabine and dFdU. Deproteinized plasma is vortexed, centrifuged, and 25 μL of the acidified extract sample is injected onto a Waters Spherisorb 4.6 mm × 250 mm, 5 μm C18 column at 40 °C. The mobile phase (flow rate, 1.0 mL/min) consists of 10:90 (v/v) acetonitrile-aqueous buffer (50 mM sodium phosphate and 3.0 mM octyl sulfonic acid, pH 2.9). Gemcitabine, dFdU, and the internal standard, 2′-deoxycytidine (2′dC) were detected with UV wavelength set at 267 nm. The standard curves for gemcitabine in both matrices ranged from 2 to 200 μM, and for dFdU in plasma, from 2 to 100 μM. Within-run and between-run component precision (CV%) was ≤6.1 and 5.7%, respectively for both human plasma and tissue culture media, and for dFdU, 2.3 and 2.7%. Total accuracy ranged from 98.7 to 106.2% for human plasma and from 96.9 to 99.2% for tissue culture media, respectively, and for dFdU, from 96.5 to 99.6%. Tetrahydrouridine (THU), an inhibitor of cytidine deaminase is used to prevent breakdown in human plasma. With one method we can measure gemcitabine in both plasma and tissue culture media. Utility is demonstrated by evaluation of the disposition of gemcitabine in an in vitro bioreactor cell culture system.
Keywords: HPLC; Gemcitabine; dFdU; In vitro bioreactor cell culture; UV;
Author Index (143-144).
Keyword Index (145-148).