Development and validation of a new gas flame ionization detector method for the determination of finasteride in tablets

A simple, rapid, and sensitive method based on gas chromatography with flame ionization detection is described for the determination of finasteride in tablets. The method is based on the derivatization of finasteride with N,O-bis(trimethylsilyl)trifluoroacetamide-1% trimethylchlorosilane at 60 degrees C for 30 min. The method was validated for specificity, linearity, precision, accuracy, robustness, and limit of quantification. The degree of linearity of the calibration curves, the percentage recoveries of finasteride, and the limit of detection (LOD) and limit of quantification (LOQ) for the gas chromatographic method were determined. The assay was linear over the concentration range of 10 to 50 microg ml(-1) (R approximately 0.999). LOQ and LOD (signal/noise ratio = 10) were found to be 10 and 2 microg ml(-1), respectively. The method was found to be simple, specific, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range. The developed method was applied successfully to estimate the amount of finasteride in tablets. The results were compared statistically with those obtained by the official method using t and F tests. There was no significant difference between the two methods with respect to mean values and standard deviations at the 95% confidence level.     

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.     

Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O(6)-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry

This work describes the determination of N3-methyladenine, N7-methylguanine and O(6)-methylguanine adducts in dimethyl sulphate-treated salmon-testes DNA employing reversed-phase high performance liquid chromatography (RP-HPLC) with UV-vis detection, followed by mass-spectrometric verification using electrospray ionisation in positive mode ESI(+). Within validation parameters, accuracy, precision, calibration parameters, limit of detection (LOD) and quantitation (LOQ) as well as stability of standard stock solutions were tested and presented for UV/vis detection. The limit of detection (LOD) was found to be 0.1 ng/mL for N3-methyladenine and 0.2 ng/mL for both N7-methylguanine and O(6)-methylguanine (S/N=3). The limit of quantitation (LOQ) was found to be 0.5 ng/mL for all measured compounds, (S/N=10). Quantitative results were obtained for each substance based on eight-point calibration. Intra- and inter-day precisions were within 1.73-6.96 and 2.26-7.58%, respectively, and correlation coefficients of calibration curves (R(2)) ranged from 0.9992 to 0.9997. Relative proportion of N7-methylguanine was accounted for 61.53+/-2.97% (R.S.D.=4.8), N3-methyladenine for 38.19+/-2.99% (R.S.D.=9.6) and O(6)-methylguanine for 0.29+/-0.02% (R.S.D.=5.1), respectively. The application of the above-mentioned techniques provides a valuable contribution for simultaneous determination of methylated DNA adducts, and may represent a suitable approach for similar monitoring/screening studies.     

Comparison of different approaches for evaluation of the detection and quantitation limits of a purity method: a case study using a capillary isoelectrofocusing method for a monoclonal antibody

Several different techniques suggested by the International Conference on Harmonization (ICH) Q2R1 guideline were used to assess the signal and concentration at the limit of detection (LOD) and limit of quantitation (LOQ) for a purity method. These approaches were exemplified with a capillary isoelectrofocusing (cIEF) method, which has been developed to quantify the distribution of the charge isoforms of a monoclonal antibody. The charge isoforms are the result of incomplete posttranslational processing of C-terminal lysine residues of the heavy chain by carboxypeptidase. Results showed no significant discrepancy between LOD/LOQ obtained by the different techniques. Validation experiments corroborated the calculated LOQ. The results indicate that any single technique can provide meaningful values for the LOD and LOQ. Finally, important points to consider when applying these techniques to purity methods are discussed.     

Determination of 8-iso-prostaglandin F(2alpha) in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.     

Simple GC-FID method development and validation for determination of alpha-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of alpha-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of alpha-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. alpha-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min(-1). Calibration curves were linear over the concentration range 1-30 microg ml(-1) (for standard solutions and solutions without endogenous alpha-tocopherol in plasma) and 5-34 microg ml(-1) (for solutions with endogenous alpha-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of alpha-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 microg.ml(-1) and 0.30 microg.ml(-1), respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of alpha-tocopherol. The endogenous alpha-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Development and validation of a gas chromatography/ion trap-mass spectrometry method for simultaneous quantification of cocaine and its metabolites benzoylecgonine and norcocaine: application to the study of cocaine metabolism in human primary cultured renal cells

Acute renal failure is a common finding in cocaine abusers. While cocaine metabolism may contribute to its nephrotoxic mechanisms, its pharmacokinetics in kidney cells is hitherto to be clarified. Primary cultures of human proximal tubular cells (HPTCs) provide a well-characterized in vitro model, phenotypically representative of HPTCs in vivo. Thus, the present work describes the first sensitive gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) using a primary culture of HPTCs as cellular matrix, following solid phase extraction (SPE) and derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The application of this methodology also enables the identification of two other cocaine metabolites: ecgonine methyl ester (EME) and anhydroecgonine methyl ester (AEME). The validation of the method was performed through the evaluation of selectivity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantification (LOQ). Its applicability was demonstrated through the quantification of cocaine, BE and NCOC in primary cultured HPTCs after incubation, at physiological conditions, with 1 mM cocaine for 72 h. The developed GC/IT-MS method was found to be linear (r2 > 0.99). The intra-day precision varied between 3.6% and 13.5% and the values of accuracy between 92.7% and 111.9%. The LOD values for cocaine, BE and NCOC were 0.97±0.09, 0.40±0.04 and 20.89±1.81 ng/mL, respectively, and 3.24±0.30, 1.34±0.14 and 69.62±6.05 ng/mL as LOQ values.     

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.     

Development and validation of isomer specific RP-HPLC method for quantification of cefpodoxime proxetil

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 degrees 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.     

Ion-pair reversed-phase HPLC: assay validation of sodium tanshinone IIA sulfonate in mouse plasma

Sodium tanshinone IIA sulfonate (STS), a hydrophilic ionic substance, is used as a cardiovascular drug. An ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) method for the determination of STS in mouse plasma was initially developed. The assay involved a rapid and simple extraction process and subsequent detection at 271 nm. The retention time for STS was 7.5 min. Based on extracted STS standard mouse plasma at 1.5,10 and 50 microg/ml, the assay precision were 2.7, 2.1 and 1.7% with a mean accuracy of 96.7, 98.5 and 99.4%, respectively. At plasma concentration of 1.5, 50 and 75 microg/ml, the mean recovery of STS were 93.1, 96.3 and 97.5%. The limit of detection (LOD) and limit of quantification (LOQ) for STS was 0.1 microg/ml and 0.5 microg/ml, respectively. Linear responses were observed over a wide concentration range (0.5-100 microg/ml) for STS in mouse plasma. STS can be detected after intravenous administration. This method was performed for the first time in pharmacokinetic studies of STS in the mouse.     

Advanced fibre optical scanning in thin-layer chromatography for drug identification

A reliable and sensitive method was developed for determination of thymol in human plasma by automated headspace solid-phase microextraction (SPME). After enzymatic cleavage of thymol sulfate thymol was extracted by a 65 microm polydimethylsiloxane-divinylbenzene crimped fiber (Supelco) after addition of sodium chloride and phosphoric acid (85%). Desorption of the fiber was performed in the injection port of a gas chromatograph at 220 degrees C (HP 5890; 50 m x 0.2 mm I.D., 0.2 microm HP Innowax capillary column; flame ionization detection). Fibers were used repeatedly up to 40 analysis. The recovery was 5% after 35 min of extraction. The calibration curve was linear in the range of 8.1-203.5 ng ml(-1) with a limit of quantitation (LOQ) of 8.1 ng ml(-1). The within-day and between-day precision and accuracy were < or = 20% at the LOQ and <15% at higher concentrations according to international guidelines for validation of bioanalytical methods. After administration of a thymol-containing herbal extract only thymol sulfate, no free thymol, could be detected in human plasma, thus analysis of thymol was after enzymatic cleavage of thymol sulfate. It is concluded that the newly developed automated method can be used in clinical trials on bioavailability and pharmacokinetics of thymol-containing herbal medicinal products.     

Measurement of anti-cancer agent methoxyamine in plasma by tandem mass spectrometry with on-line sample extraction

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis HLB extraction column (2.1 mm x 20 mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00-1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1-94.7 and 90.1-96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse.     

Rapid analytical method for the determination of 220 pesticide with their isomers by GCMS-TIC

This paper presents a cost-effective and validated multi residue modified and miniaturized method for the determination of 220 chemically different groups of pesticides and their isomers. This determination method is performed with single Quaid Gas Chromatography Mass Spectrometry -Total Ion Chromatogram GCMS-TIC. Two methods was experimented and modified with different GCMS parameters to analyses most common used pesticide and their residues in the standers solution and can be applied for real environmental samples. The results showed by single Quaid GCMS-TIC it can analyze 220 pesticides including their isomers within 49.6 min and low detection limit by using modified method 2 as described in this research. Limit of detection (LOD) was ranged from 0.78 to 14.74 ng/ml (ppb) with good separation and resolution. Limit of quantification (LOQ) was ranged between 2.34 and 44.22 ng/ml (ppb). Method 2 was more accurate, shorter, and clear separation rather than method 1. This method can be successfully applied in real environmental samples proven to be a good option for routine analysis of pesticide within the maximum residue limits (MRL) referenced to European commission especially with the most common GCMS-TIC which exists in most of labs and low income countries.     

Determination of methylnaltrexone in clinical samples by solid-phase extraction and high-performance liquid chromatography for a pharmacokinetics study

A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 microm, 150x3.2 mm analytical column and 50 mM potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit (r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% (n = 10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C(max) = 110 (+/-55) and 287 (+/-101) ng/ml and t(max) = 16.7 (+/-10.8) and 20.0 (+/-9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.     

Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection. Preliminary validation of the method performance with spiked (0.033-1.0 ng OTA/ml) and commercial red wines provided recoveries >90% and < 10%, with limit of detection (LOD) and limit of quantification (LOQ) of 0.01 and 0.033 ng/ml. However, a similarly favorable performance characteristics was observed in control experiments in which the MIP was replaced by the corresponding non-imprinted polymer (NIP). These findings provide evidence that under the employed experimental conditions specific analyte binding to imprinted binding sites plays a minor role in selective OTA retention. In the framework of this study, other problems inherent to MIP-based solid-phase extraction have been addressed. These include the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavorable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding, and slow analyte binding kinetics.     

Bioanalysis of tobramycin for therapeutic drug monitoring by solid-phase extraction and capillary zone electrophoresis

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis (CZE) for the analysis of tobramycin in human serum is presented. An off-line SPE employing a carboxypropyl bonded phase (CBA) cartridge was used for the extraction of tobramycin from human serum. Adsorbed tobramycin was eluted from the CBA cartridge using a mixture of NH(3) (25%, w/v)-methanol (30:70, v/v). After evaporation, the analyte was reconstituted and derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA). The resulting tobramycin-OPA/MPA derivative was purified, and then identified by mass spectrometry. The tobramycin-OPA/MPA derivative was then analysed by CZE with a background electrolyte (BGE) comprising of 30 mM sodium tetraborate pH 10.0-acetonitrile (ACN) (80:20, v/v) with ultraviolet detection at 230 nm. A linear response was observed in the range of 0.3-30 microg/ml with r(2) = 0.992. The sensitivity of the method was determined by its limit of quantitation (LOQ) and limit of detection (LOD) of 0.3 microg/ml and 0.1 microg/ml, respectively. SPE recovery ranged from 68 to 79% at the trough levels to 98% at the peak levels found in serum. Furosemide has been added as internal standard (IS) to improve precision. For the therapeutic range of tobramycin in serum (2-10 microg/ml) the relative standard deviation (R.S.D.) was less than 11% for the entire SPE/CE process. The method demonstrated excellent selectivity as shown by the lack of interference from a total of 20 drugs investigated. The method was then used in therapeutic drug monitoring of patients receiving the drug.     

Rapid determination of 5-fluorouracil in plasma using capillary electrophoresis

A rapid, simple and sensitive capillary electrophoresis (CE) method used for the determination of 5-fluorouracil in rabbit plasma is described in the present paper. In this method, samples were simply pretreated by a solvent extraction procedure prior to injection. With a running buffer composed of 30 mM Tris-H(3)PO(4) (pH 7.0) and 5% isopropanol, 5-fluorouracil was easily separated from the external standard alpha-phenethylol as well as other substances existed in the plasma. A linearity of 5-fluorouracil was determined in the range from 0.17 to 42.50 microg/ml with a correlation coefficient of 0.999. A limit of quantitation (LOQ) corresponding to signal-to-noise ratio of 10 was obtained (LOQ=0.08 microg/ml). The method was successfully used for determining the 5-fluorouracil in real plasma samples from rabbits.     

Quantifying crystal form content in physical mixtures of (±)-tartaric acid and (+)-tartaric acid using near infrared reflectance spectroscopy

The objective of this study was to use diffuse reflectance near infrared spectroscopy (NIRS) to determine racemic compound content in physical mixtures composed primarily of the enantiomorph and to assess the error, instrument reproducibility and limits of detection (LOD) and quantification (LOQ) of the method. Physical mixtures ranging from 0 to 25% (±)-tartaric acid in (+)-tartaric acid were prepared and spectra of the powder samples contained in glass vials were obtained using a Foss NIRSystems Model 5000 monochrometer equipped with a Rapid Content Analyzer scanning from 1100 to 2500 nm. A calibration curve was constructed by plotting (±)-tartaric acid weight percent against the 2^nd derivative values of log (1/R) vs λ at a single wavelength, normalized with a denominator wavelength (1480 nm/1280 nm). Excellent linearity was observed (R²=0.9999). The standard error of calibration (SEC) was 0.07 and the standard error of prediction (SEP) for the validation set was 0.11. Instrument and method errors for samples in the 2% composition range ((±)-tartaric acid in (+)-tartaric acid) were less than 1% RSD and 3% RSD, respectively. The practical LOD and LOQ were 0.1% and 0.5%, respectively, and comparable to the calculated LOD and LOQ. These studies show that NIRS can be used as a rapid and sensitive quantitative method for determining racemate content in the presence of the enatiomerically pure crystal in the solid-state. Published: October 6, 2005     

Simultaneous determination and pharmacokinetic studies of dihydromyricetin and myricetin in rat plasma by HPLC-DAD after oral administration of Ampelopsis grossedentata decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination of dihydromyricetin (1) and myricetin (2) in rat plasma after orally administrating the decoction of Ampelopsis grossedentata. Plasma samples were acidified with 0.375% phosphoric acid and extracted with ethyl acetate. Analysis of the extract was performed on reversed-phase C(18) column with a gradient eluent composed of acetonitrile and 0.04% phosphoric acid. The flow rate was kept at 1 ml/min and the detection wavelength was set at 290 and 370 nm for 1 and 2, respectively. The calibration curves were linear in the range of 0.247-4.114 microg/ml and 0.150-2.501 microg/ml for 1 and 2, respectively. The intra-day and inter-day precisions were better than 4.9 and 6.2%, respectively. The limits of detection (LOD) for 1 and 2 in plasma were 21.600 and 52.530 ng/ml, and the limits of quantification (LOQ) were 0.247 and 0.150 microg/ml, respectively. The mean recoveries for 1 and 2 were 92.0 and 93.3%, respectively. The accuracy and precision were well within the acceptable range and R.S.D. of measured rat samples was less than 7.5%. This validated method has been successfully applied in the pharmacokinetics study of dihydromyricetin and myricetin in vivo after orally administrating the decoction of A. grossedentata to rats.