Stability studies of anticancer agent bis(4-fluorobenzyl)trisulfide and synthesis of related substances

Bis(4-fluorobenzyl)trisulfide, fluorapacin, has been extensively developed as a promising new anticancer drug candidate. Its degradation products were identified and verified by the newly synthesized compounds bis(4-fluorobenzyl)disulfide (A) and bis(4-fluorobenzyl)tetrasulfide (B) which were resulted from the disproportionation of fluorapacin under forced conditions. A stability-indicating HPLC method was used for the stability evaluation of active pharmaceutical ingredient (API) fluorapacin and finished pharmaceutical product (FPP) under various conditions. High recovery (99.57%) of API was found after three freeze-thaw cycle processes of fluorapacin FPP. Susceptibility of fluorapacin to oxidative degradation was studied by treating fluorapacin and FPP in 30% hydrogen peroxide aqueous solution, and the result verified the oxidative stability of fluorapacin. However, treatment of this drug candidate under strong light (4500 Lx+/-500 Lx) for 10 days showed substantial effect on the recovery of fluorapacin, especially from fluorapacin FPP. Strong acid (1.0M, HCl) did not affect the recovery of fluorapacin while strong basic condition (1.0M, NaOH) accelerated the disproportionation of fluorapacin to its related substances A and B. The stability of fluorapacin in its aqueous media at a pH range of 2.0-10.0 for up to 6h was further investigated, and 4.0-8.0 was found to be the most stable pH range. Fluorapacin and FPP were exposed to the elevated temperatures of 40 and 60 degrees C for 10 days without obvious impact on their stability. The thermal stability of fluorapacin API and FPP under constant humidity with light protection was also thoroughly investigated under accelerated (40+/-2 degrees C, RH 75+/-5%, 6 months) and long-term (25+/-2 degrees C, RH 60+/-10%, 24 months) conditions. There was no significant change except minor color change of fluorapacin FPP. Therefore, fluorapacin has excellent stability as a potential drug candidate for further clinical development investigation.     

Quantitative determination and sampling of azathioprine residues for cleaning validation in production area

Cleaning validation is an integral part of current good manufacturing practices in any pharmaceutical industry. Nowadays, azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9 mm x 150 mm, 4 microm) and methanol-water-acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0 mL min(-1). UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 microg mL(-1) with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit (QL) were 0.09 and 0.29 microg mL(-1), respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. Nevertheless, the simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.     

LC method for the analysis of Oxiconazole in pharmaceutical formulations

A LC method has been developed for the quantitative determination of Oxiconazole (Ox) in bulk form, lotion and cream pharmaceutical formulations. The method validation yielded good results including the range, linearity, precision, accuracy, recovery, specificity, robustness, limit of quantitation and limit of detection. The LC separation was carried by reversed phase chromatography using a LiChrocart C(8) column (125 mm x 4.0 mm i.d., 5 microm particle size). The mobile phase was composed of methanol-0.02 M ammonium acetate buffer (85:15 v/v), pumped isocratically at flow rate 1 ml min(-1). The detection was carried out on UV detector at 254 nm. The calibration curve for Ox was linear from 40.0-140.0 microg ml(-1) range. The precision of this method, calculated as the relative standard deviation (R.S.D.) was 1.57% for lotion and 0.71% for cream. The R.S.D. values for intra- and inter-day precision studies were 0.57 and 1.34%, respectively. The recovery of the drug ranged between 98.84-102.2% (lotion) and 100.54-101.59% (cream). The stability indicating capability of the assays was proved using forced degradation. Chromatograms showed Ox well resolved from the degradation product.     

Quantification of levetiracetam in human plasma by liquid chromatography-tandem mass spectrometry: application to therapeutic drug monitoring

A rapid, selective, reliable, precise, accurate, and reproducible tandem mass spectrometric (MS-MS) method for the quantification of levetiracetam (LEV) in human plasma using adenosine as an internal standard (IS) has been developed and validated. The drug and IS were extracted by solid phase extraction (SPE) technique and analyzed on Symmetry((R)) C(18) column (5 microm, 3.9 mm x 50 mm) using a mobile phase of methanol-water-formic acid (97:03:0.25, v/v/v) at a flow rate of 0.2 ml/min. Quantitation was achieved using a positive electrospray ionization (ESI+) interface employing multiple reaction monitoring (MRM) mode at MRM transitions m/z 171>126 and m/z 268>136 for LEV and IS, respectively. The method was validated over the concentration range of 1.0-40 microg/ml (r>0.99) with a limit of quantification of 1.0 microg/ml (R.S.D.%; 4.1 and Bias%; -9.0 to + 11.0%). Intra- and inter-run precision of LEV assay at three concentrations ranged from 0.6 to 8.9% with accuracy (bias) varied from -4.0 to 8.6% indicating good precision and accuracy. Analytical recoveries of LEV and IS from spiked human plasma were in the range of 91.7-93.4% and 80.2-84.1%, respectively. Stability of LEV in human plasma samples at different conditions showed that the drug was stable under the studied conditions. Matrix effect study showed a lack of matrix effect on mass ions of LEV and IS. The described method compared well with the commercial HPLC-UV method of Chromsystem (r(2)=0.99). The suitability of the developed method for therapeutic drug monitoring was demonstrated by measuring LEV in human plasma samples of epileptic patients treated with LEV.     

HPLC determination of sertraline in bulk drug, tablets and capsules using hydroxypropyl-beta-cyclodextrin as mobile phase additive

A sensitive and stereospecific high-performance liquid chromatography (HPLC) method for determination of sertraline in bulk drug, tablets and capsules was developed. Chromatography resolution of the sertraline enantiomeric forms and trans diastereoisomers was performed on Alltima C18 (250 mm x 4.6mm i.d., 5 microm) column with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as mobile phase additive. The composition of the mobile phase was 68:32 (v/v) aqueous 170 mM phosphate buffer, pH 3.0 (adjusted with 85% phosphoric acid) containing 18 mM HP-beta-CD/acetonitrile at a flow rate of 1.0 ml x min(-1). The UV detector was set at 225 nm. Calibration curves were linear (r=0.9999, n=9) in the range of 1-120 microgml (-1) for sertraline. Limit of detection and quantitation for sertraline was 0.029 and 0.097 microg x ml (-1). The values of R.S.D. of repeatability and intermediate precision for bulk drug, tablets and capsules of sertraline hydrochloride were less than 1.0%.     

Quantitative determination of azithromycin in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of azithromycin in human plasma was developed and validated. Azithromycin in plasma (0.2mL) was extracted with methyl tert-butyl ether-hexane (50:50, v/v), organic phase was transferred to another clear 1.5mL Eppendorf tube and evaporated to dryness at 40 degrees C and dissolved in mobile phase, samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150mmx2.1mm i.d., 5microm), together with a mobile phase containing of 20mM ammonium acetate (pH 5.2)-acetonitrile-methanol (50:40:10, v/v/v) and was isocratically eluted at a flow rate of 0.2mL/min. Azithromycin and its internal standard, clarithromycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 2 to 1000ng/mL with r=0.9977. The limit of quantification for azithromycin in plasma was 2ng/mL with good accuracy and precision. The higher mean extraction recovery, say 81.2% and 75.5% for azithromycin and internal standard (IS), respectively, was obtained in this work. The intra-day and inter-day precision ranged from 4.8% to 8.6% and 6.4% to 10.7% (R.S.D.), respectively. The established method has been successfully applied to bioequivalence study of 2 azithromycin formulations for 24 healthy volunteers.     

RP-HPLC determination of recombinant human interferon omega in the Pichia pastoris fermentation broth

A rapid and valid reversed-phase high performance liquid chromatography (RP-HPLC) method for determination of recombinant human interferon omega (rhIFNomega) in the yeast Pichia pastoris fermentation broth was developed. The method is based on the hydrophobicity of rhIFNomega followed by RP-HPLC separation with UV detection. The chromatography analysis was performed on EC 250/4 NUCLEOSIL 300-5 C18 (250 mm x 4 mm i.d., 300 A, with a particle size of 5 microm) column. The compositions of the mobile phase A and B were 999:1 (v/v) water/TFA and 999:1 (v/v) acetonitrile/TFA at a flow rate of 1.0 ml min(-1). Detection was done by spectrophotometry at 280 nm and the column temperature was 30+/-1 degrees C. Calibration curve was linear (r=0.9986, n=7) in the range of 0.074-0.555 mg ml(-1) for rhIFNomega and the regression equation was y=2.02 x 10(6)x-1.27 x 10(5). Limit of detection for rhIFNomega was 0.053 mg ml(-1). The values of R.S.D. (%) of intra-day and inter-day precision were <5.65 and <5.68 (n=6), respectively. The R.S.D. (%) values and the average recovery rate of recovery experiment were <1.23 (n=3) and 97.97%.     

Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry

High turbulence liquid chromatography (HTLC, or turbulent flow online extraction) and tandem mass spectrometry (MS/MS) methods for the determination of sitagliptin in human urine and hemodialysate were developed and validated to support clinical studies. A narrow bore large particle size reversed-phase column (Cyclone, 50 mm x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 mm x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. For the urine assay, the LLOQ was 0.1 microg/ml, the linear calibration range was 0.1 to 50 microg/ml, the interday precision (R.S.D.%, n=5) was 2.3-6.5%, and the accuracy was 96.9-106% of the nominal value. For the urine quality control samples (QCs), the intraday precision (R.S.D.%, n=5) and accuracy were 1.8-2.6% and 96.2-106% of the nominal value, respectively. The interday precision (R.S.D.%) for 56 sets of urine QCs over a 6-month period varied from 3.8% to 5.5% and the accuracy from 102% to 105% of the nominal value. For the hemodialysate assay, the LLOQ was 0.01 ng/ml, the linear dynamic range was 0.01-5.0 ng/ml, the interday precision was 1.6-4.1%, and the accuracy was 89.8-104% of the nominal value. For hemodialysate QCs, the intraday precision and accuracy varied from 2.3% to 8.9% and from 99.8% to 111% of the nominal value, respectively. These results demonstrated that both methods are selective, accurate, precise, reproducible, and suitable for quantifying sitagliptin in hemodialysate and human urine samples.     

An improved and validated LC method for resolution of bicalutamide enantiomers using amylose tris-(3,5-dimethylphenylcarbamate) as a chiral stationary phase

An improved HPLC method for determination of enantiomeric purity of bicalutamide in drugs and pharmaceuticals was developed and validated. Baseline separation with resolution >/=6.0 was achieved within 10 min on Chiralpak AD-H (250 mm x 4.6 mm; particle size 5 microm) column using n-hexane:2-propanol (65:35 v/v) as mobile phase at a flow rate of 1.0 ml/min at 25 degrees C. The detection was made at 270 nm using UV detector while a polarimetric detector connected in series was used for identification of enantiomers. The effects of 2-propanol, ethanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was validated in terms of accuracy, precision and linearity in the range of 10-250 microg/ml and the r(2) was >0.9999. The recoveries were 99.68-100.25% with <1% R.S.D. The limits of detection (LOD) and quantification (LOQ) of enantiomers were (2.4, 3.0 and 7.6, 9.3) x 10(-8)g/ml for (S)-(+)-BCT and (R)-(-)-BCT enantiomers, respectively. The method was found to be suitable for rapid determination of enantiomeric purity of bicalutamide in bulk drugs and pharmaceutical formulations.     

Simultaneous determination of warfarin enantiomers and its metabolite in human plasma by column-switching high-performance liquid chromatography with chiral separation

A simple and sensitive column-switching high-performance liquid chromatographic method for the simultaneous determination of warfarin enantiomers and their metabolites, 7-hydroxywarfarin enantiomers, in human plasma is described. Warfarin enantiomers, 7-hydroxywarfarin enantiomers, and an internal standard, diclofenac sodium, were extracted from 1 mL of a plasma sample using diethyl ether-chloroform (80:20, v/v). The extract was injected onto column I (TSK precolumn BSA-C8, 5 microm, 10 mm x 4.6 mm inside diameter) for cleanup and column II (Chiralcel OD-RH analytical column, 150 mm x 4.6 mm inside diameter) coupled with a guard column (Chiralcel OD-RH guard column, 10 mm x4.6 mm inside diameter) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (84:16 v/v, pH 2.0) for clean-up and phosphate buffer-acetonitrile (45:55 v/v, pH 2.0) for separation. The peaks were monitored with an ultraviolet detector set at a wavelength of 312 nm, and total time for chromatographic separation was approximately 25 minutes. The validated concentration ranges of this method were 3 to 1000 ng/mL for (R)- and (S)-warfarin and 3 to 200 ng/mL for (R)- and (S)-7-hydroxywarfarin. Intra- and interday coefficients of variation were less than 4.4% and 4.9% for (R)-warfarin and 4.8% and 4.0% for (S)-warfarin, and 5.1% and 4.2% for (R)-7-hydroxywarfarin and 5.8% and 5.0% for (S)-7-hydroxywarfarin at the different concentrations. The limit of quantification was 3 ng/mL for both warfarin and 7-hydroxywarfarin enantiomers. This method was suitable for therapeutic drug monitoring of warfarin enantiomers and was applied in a pharmacokinetic study requiring the simultaneous determination of warfarin enantiomers and its metabolite, 7-hydroxywarfarin enantiomers, in human volunteers.     

Quantitative determination of gatifloxacin, levofloxacin, lomefloxacin and pefloxacin fluoroquinolonic antibiotics in pharmaceutical preparations by high-performance liquid chromatography

The objective of this research was to develop and validate analytical methods for quantitative determination of fluoroquinolones of third generation. Simple and rapid chromatographic method was developed and validated for quantitative determination of four quinolone antibiotics in tablets and injection preparations. The fluoroquinolones studied were gatifloxacin (GAT), levofloxacin (LEV), lomefloxacin (LOM) and pefloxacin (PEF). The quinolones were analyzed by using a LiChrospher 100 RP-18 column (5 microm, 125 mm x 4 mm) and a mobile phase constituted of water:acetonitrile (80:20, v/v) with 0.3% of triethylamine and pH adjusted to 3.3 with phosphoric acid. The flow rate was 1.0 mL/min and the analyses were performed using UV detector with wavelengths varying from 279 to 295 nm. The analyses were performed at room temperature (24 +/- 2 degrees C). All fluoroquinolones were separated within 5 min. The calibration curves were linear (r>or=0.9999) over a concentration range from 4.0 to 24.0 microg/mL. The relative standard deviation (R.S.D.) was < 1.0% and average recovery was above 99.54%.     

Quantitative determination of amantadine in human plasma by liquid chromatography-mass spectrometry and the application in a bioequivalence study

A sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method is developed and validated for rapid determination of amantadine in human plasma. Desloratadine was used as the internal standard (I.S.). Human plasma (0.2 mL) was first alkalified with 100 microL of sodium hydroxide (3M) and then extracted with 1 mL of n-hexane containing 1% isopropanol (v/v) and 10% dichloromethane (v/v) by vortex-mixer for 3 min. The mixture was centrifuged at 14,000 rpm for 5 min. The supernatant was evaporated to dryness and the residue was dissolved in mobile phase. Samples were separated using a Thermo Hypersil-HyPURITYC18 reversed-phase column (150 mm x 2.1 mm i.d., 5 microm). Mobile phase consisted of methanol-acetonitrile-20 mM ammonium acetate (45:10:45, v/v/v) containing 1% acetic acid with pH 4.0. Amantadine and I.S. were measured by electrospray ion source in positive selective ion monitoring mode. The good linearity ranged from 3.9 to 1000 ng/mL and the lowest limit of quantification was 3.9 ng/mL. The extraction efficiencies were approximately 70% and recoveries of method ranged from 98.53 to 103.24%. The intra-day relative standard deviations (R.S.D.) were less than 8.43% and inter-day R.S.D. below 10.59%. The quality control samples were stable when kept at room temperature for 12h, at -20 degrees C for 30 days and after four freeze/thaw cycles. The method has been successfully used to evaluation of the pharmacokinetics and bioequivalence of amantadine in 20 healthy volunteers after an oral dose of 100 mg amantadine.     

Simultaneous determinations of paeonol and palmatine hydrochloride in Shangshi Aerosols by HPLC method

A simple and rapid HPLC method was described for the simultaneous determination of paeonol and palmatine hydrochloride in Shangshi Aerosols. The optimum separation for these analytes was achieved using the mixture of 0.025 M sodium dihydrogen phosphate-acetonitrile-diethylamine (64:35:1, v/v/v) as the mobile phase and a Nova-Pak((R)) C8 column. The linear ranges of paeonol and palmatine hydrochloride were 0.2-80 and 0.06-60 microg/ml with the regression equations being Y=11716.4+2.96 x 10(6)X (R=0.99969), Y=-6388.8+1.89 x 10(5)X (R=0.99976), and limit of quantifications (LOQ) for paeonol and palmatine hydrochloride were 0.2 and 0.06 microg/ml, respectively (n=6). Other validation parameters: intra-day precision (R.S.D.: 0.71-1.65%) and inter-day precision (R.S.D.: 0.89-2.11%), and reproducibility (recoveries values: 94.6-98.2% for paeonol, 94.85-97.58% for palmatine hydrochloride) were found to be satisfactory. The proposed HPLC method had been applied for the determination of paeonol and palmatine hydrochloride in Shangshi Aerosols; R.S.D. values were 1.45 and 1.13%, respectively. In short, this method was rapid and convenient, which could be used for the routine control of paeonol and palmatine hydrochloride in Shangshi Aerosols.     

Analysis of naphthoquinone derivatives in the Asian medicinal plant Eleutherine americana by RP-HPLC and LC-MS

The first analytical procedure for the determination of a new naphthopyrone, eleutherinoside A, together with the known bioactive compounds eleuthoside B, isoeleutherin, eleutherin and eleutherol in Eleutherine americana was established. Optimum HPLC separation of these naphthoquinone derivatives was possible on RP-12 column material, using water and acetonitrile as mobile phase. Flow-rate, detection wavelength and temperature were adjusted to 1.0 mL/min, 254 nm and 40 degrees C, respectively. Validation results indicated that the HPLC method is well suited for the determination of naphthoquinone derivatives in the bulbs of E. americana with a good linearity (r2>0.9996), precision (intra-day R.S.D. <4.70%, inter-day R.S.D. <5.68%) and recovery rates from 96.26 to 103.48%. Limit of detection (LOD) was found to be below 0.84 microg/mL for all five compounds. LC-MS analyses performed in positive and negative electrospray ionization mode assured peak purity and identity. The analysis of different E. americana samples from Thailand revealed that eleutherol (0.10-0.20%) was dominant in all specimens, followed by isoeleutherin and eleutherin. The new natural product 2,5-dimethyl-10-hydroxynaphthopyrone 8-O-beta-d-glucopyranoside occurred in percentages of less than 0.05%.     

Simultaneous determination of ranitidine and metronidazole in human plasma using high performance liquid chromatography with diode array detection

The development and validation of a simple method for the simultaneous determination of ranitidine and metronidazole in human plasma is described. Plasma samples (250 microL) were deproteinized by precipitation with 60% perchloric acid, centrifuged and the supernatant directly injected into the HPLC. Separation was achieved in isocratic mode with a Shimpak C(18) column and a mobile phase consisting of 10mM potassium dihydrogen phosphate pH 3.5:acetonitrile (90:10, v/v) with UV detection at 315 nm. The method showed good selectivity and sensitivity. Good and consistent recovery for metronidazole and ranitidine was obtained: 96.22+/-3.52 and 95.00+/-4.50% for ranitidine (25-1000 ng/mL) and metronidazole (60-10,000 ng/mL), respectively (n=3). With this one-step sample preparation method, both ranitidine and metronidazole could be quantified simultaneously in human plasma with good precision (R.S.D.<15%) and accuracy (bias values below 15%). The limit of quantification for ranitidine and metronidazole were 20 and 40 ng/mL plasma, respectively.     

A validated RP-HPLC method for quantitative determination of related impurities of ursodeoxycholic acid (API) by refractive index detection

An isocratic RP-HPLC method was developed and validated for quantitative determination of ursodeoxycholic acid (UDCA) and its related impurities. Considering the lower molecular absorptivity of UDCA, refractive index detector was used to detect the impurities on a Phenomenex Luna C(18), 150 mm × 4.6 mm, 5 μm column. The mobile phase was 0.1% acetic acid/methanol (30:70, v/v) and flow rate was 0.8 ml/min. The detector and column temperature was maintained at 40°C. The method is linear over a range of 0.25-3.5 μg/ml for all impurities and coefficient of correlation (r(2)) was ≥0.9945. The accuracy of method demonstrated at three levels in the range of 50-150% of the specification limit and recoveries were found to be in the range of 97.11-100.75%. The precision for all related impurities was below 3.5% R.S.D. The method was applied to commercial bulk drug sample for assay purpose.     

Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a clinical study

A simple, sensitive and specific LC-MS/MS method for simultaneous determination of rosuvastatin (RST) and fenofibric acid (FFA) was developed and validated with 500 microL human plasma using carbamazepine as an internal standard (IS). The assay procedure involved a simple one-step liquid/liquid extraction of RST and FFA and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto X-Terra MS C-18 column (4.6 mm x 50 mm, 5.0 microm). Separation of RST, FFA and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (45:55, v/v) at a flow rate of 0.40 ml/min. The API-3000LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Positive ion acquisition chromatographic run was used in the present method. Nominal retention times of RST, FFA and IS were 2.35, 4.70 and 2.32 min, respectively. Absolute recovery of RST, FFA and IS was 74, 61 and 69%, respectively. The lower limit of quantification (LLOQ) of RST and FFA was 1.00 ng/ml and 0.50 microg/ml, respectively. Response function was established for the range of concentrations 1.00-50.0 ng/ml and 0.50-20.0 microg/ml for RST and FFA, respectively, with a coefficient of determination (r2) of 0.999 for both the compounds. The inter- and intra-day precision in the measurement of RST quality control (QC) samples 5, 15, 400 and 800 ng/ml, were in the range 8.93-9.37% relative standard deviation (R.S.D.) and 1.74-16.1% R.S.D., respectively. Similarly, the inter- and intra-day precision in the measurement of FFA quality control (QC) samples 0.5, 1.5, 8.0 and 15.0 microg/ml, were in the range 9.78-11.6% relative standard deviation (R.S.D.) and 0.22-17.4% R.S.D., respectively. Accuracy in the measurement of QC samples for RST and FFA were in the range 88.1-108 and 87-115%, respectively, of the nominal values. RST and FFA were stable in the battery of stability studies, viz., bench-top, auto-sampler and freeze/thaw cycles. Stability of RST and FFA was established for 1 month at -80 degrees C. The application of the assay to a clinical study confirmed the utility of the assay.     

A sensitive LC/MS/MS method using silica column and aqueous-organic mobile phase for the analysis of loratadine and descarboethoxy-loratadine in human plasma

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed and validated for the simultaneous analysis of antihistamine drug loratadine (LOR) and its active metabolite descarboethoxy-loratadine (DCL) in human plasma. Deuterated analytes, i.e. LOR-d(3) and DCL-d(3) were used as the internal standards (I.S.). Analytes were extracted from alkalized human plasma by liquid/liquid extraction using hexane. The extract was evaporated to dryness under nitrogen, reconstituted with 0.1% (v/v) of trifluoroacetic acid (TFA) in acetonitrile, and injected onto a 50 x 3.0 mm I.D. 5 microm, silica column with an aqueous-organic mobile phase consisted of acetonitrile, water, and TFA (90:10:0.1, v/v/v). The chromatographic run time was 3.0 min per injection and flow rate was 0.5 ml/min. The retention time was 1.2 and 2.0 min for LOR and DCL, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transitions: 383-->337 (m/z) for LOR, 311-->259 (m/z) for DCL, 388-->342 (m/z) for LOR-d(3), and 316-->262 (m/z) for DCL-d(3). The low limit of quantitation (LLOQ) was 10 pg/ml for LOR and 25 pg/ml for DCL. The inter-day precision of the quality control (QC) samples was 3.5-9.4% relative standard deviation (R.S.D.). The inter-day accuracy of the QC samples was 99.0-107.9% of the nominal values.     

HPLC法测定罗氟司特原料药的含量

目的：建立高效液相色谱法测定罗氟司特原料药含量的分析方法。方法选用Agilent C18色谱柱（250mm ＆#215;4.6mm,5μm）;以0.005mol · L ^-1甲酸铵溶液（pH 3.5）-乙腈（40∶60,v/v）为流动相;柱温30℃;流速：1.0mL· min ^-1;检测波长：250nm。结果罗氟司特的线性关系良好（25.05～100.2μg· mL^ -1,r＝0.9998）;平均加样回收率为100.0％,RSD＝1.0％（n＝9）;检测限与定量限分别为1.7ng和5.0ng;重复性、中间精密度均符合要求。结论本方法专属性强,简捷,准确,可用于罗氟司特原料药的含量测定。     

First derivative ratio spectrophotometric,HPTLC-densitometric,and HPLC determination of nicergoline in presence of its hydrolysis-induced degradation product

Three methods are presented for the determination of Nicergoline in presence of its hydrolysis-induced degradation product. The first method was based on measurement of the first derivative of ratio spectra amplitude of Nicergoline at 291 nm. The second method was based on separation of Nicergoline from its degradation product followed by densitometric measurement of the spots at 287 nm. The separation was carried out on HPTLC silica gel F(254) plates, using methanol-ethyl acetate-glacial acetic acid (5:7:3, v/v/v) as mobile phase. The third method was based on high performance liquid chromatographic (HPLC) separation and determination of Nicergoline from its degradation product on a reversed phase, nucloesil C(18) column using a mobile phase of methanol-water-glacial acetic acid (80:20:0.1, v/v/v) with UV detection at 280 nm. Chlorpromazine hydrochloride was used as internal standard. Laboratory prepared mixtures containing different percentages of the degradation product were analysed by the proposed methods and satisfactory results were obtained. These methods have been successfully applied to the analysis of Nicergoline in Sermion tablets. The validities of these methods were ascertained by applying standard addition technique, the mean percentage recovery +/- R.S.D.% was found to be 99.47 +/- 0.752, 100.01 +/- 0.940, 99.75 +/- 0.740 for the first derivative of ratio spectra method, the HPTLC method and the HPLC method, respectively. The proposed methods were statistically compared with the manufacturer's HPLC method of analysis of Nicergoline and no significant difference was found with respect to both precision and accuracy. They have the advantage of being stability indicating. Therefore, they can be used for routine analysis of the drug in quality control laboratories.