High-Performance Liquid Chromatographic Method for the Simultaneous Assay of Reserpine, Clopamied and Dihydroergocristine Mesylate in Tablets

Objective To establish a liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitation determination of plasma imatinib mesylate and perform the general method Validation. Methods After proteins in plasma were precipitated, imatinib was analyzed by LC-MS/MS mode and multiple reaction monitor (MRM) mode, the ion pairs monitored were m/z 494.3→m/z 394.2 (imatinib), m/z237.3→m/z194.1 (carbamazepine, internal standard). Results The linear range was 6.25 to 3 200.00 μg/L. The within-run precision was 1.98%-4.46%, and between-run precision was 3.07%-7.54%. The correctness biaswas-7.34%-2.28%. Matrix effect factor was 0.93-1.05. Conclusion The method established is sensitive, accurate and stable, satisfying the quantification requirements of imatinib mesylate in plasma, and it could be used in clinical analysis. Key words: Imatinib mesylate; Liquid chromatography tandem mass spectrometry; Method validation

Objective To make comparative analysis of the glycated hemoglobin A1c(HbA1c) values obtained by three HbA1c test methods in patients with variant hemoglobins. Methods Total of 50 blood samples of patients with different types of variant hemoglobin were collected from January 2012 to December 2012; 25 of them (14 males and 11 females) carried hemoglobin D, Q, G, J and E with a mean age of (24±3) years; the other 25 cases (11 males and 14 females) were blood samples with hemoglobin F from newborn infants. Meanwhile, 50 blood samples(25 males and 25 females) from people without variant hemoglobins were also collected as control(mean age (25±5) years). Three methods were used to test HbA1c, which were affinity high performance liquid chromatography (HPLC) method (Ultra 2 of American Primus), ion exchange HPLC method (VariantⅡ of American Bio-Rad and G8 of Japanese Tosoh) and immunization method (DCA Vantage of German Siemens). The statistic analysis were done with variance analysis and correlation analysis. Results The HbA1c of the group with normal HbA1c structure was 5.7%±1.1%, 5.7%±1.2%, 5.7%±1.2% and 5.7%±1.1% respectively when it was tested by affinity HPLC method (Ultra2), ion exchange HPLC method (G8), ion exchange HPLC (Variant Ⅱ) and immunization method (DCA Vantage), and there was no significant difference among the groups (F=0.023, P>0.05). In the 25 samples with hemoglobin F, HbA1c could not be detected by ion exchange HPLC or immunoassay method. The fasting blood glucose level correlated with HbA1c level tested by Ultra 2 method (r=0.647, P<0.05), but it didn't correlate with HbA1c level when tested with VariantⅡ and G8 as well as DCA Vantage method. The HbA1c result of affinity HPLC method was free from the disturbance of Hb D, Q, G, J and E, and had a obvious correlation with blood glucose (r=0.823, P<0.05). The HbA1c result of ion exchange HPLC method was disturbed by hemoglobin D, Q, G, J and E in varying degrees. The HbA1c measured by immunization method was associated with blood glucose (r=0.611, P<0.05). Conclusion The HbA1c value obtained by affinity HPLC method can accurately reflect the mean blood glucose level. Variant hemoglobins disturb the HbA1c result tested by ion exchange HPLC method, and the immunization test result is only disturbed by hemoglobin F. Key words: Glycated hemoglobin A1c; Variant hemoglobins; High performance liquid chromatography

Liquid chromatography coupled to tandem mass spectrometry (LC-MS) has been used in the last two decades, in drug discovery or drug development as well as in therapeutic drug monitoring (TDM). Due to its high sensitivity and selectivity it is the method of choice for the quantification of drugs and metabolites in biological fluids. More and more the technique is also applied for multi-analytes quantification to support metabolomics studies. To improve sample throughput in TDM a dual Ultra High Performance Liquid Chromatography – Mass Spectrometry assay was developed and validated for the quantification of eight protease inhibitors in plasma and blood. Based on the same platform with two different chromatographic systems (pH 2.7 and pH 6.6) and the addition an automated on-line sample preparation step an assay method could be developed for the analysis of eighty-seven metabolites in plasma and blood. Finally the downsizing of the LC was investigated with the emphasis to improve the sensitivity of LC-MS assay when microsamples are available.

Hemoglobin A1c (HbA1c) measurement devices are widely used to evaluate glycemic control in diabetic patients. The aim of this study was to investigate the comparability of various HbA1c instruments used in Iran. In the present study, 154 fresh whole blood samples from diabetic patients, with different HbA1c levels (4.0%-10%) and no types of hemoglobinopathy were analyzed by six HbA1c assays including one high performance liquid chromatography (HPLC) method (D10 HbA1c), two immunoassay methods (COBAS INTEGRA 400 and Pars Azmoon kit), one Boronate affinity method (Nycocard Reader II), and two ion exchange methods (Biosystems and DS5). The two National Glycohemoglobin Standardization Programs (NGSP) certified system, D10 and COBAS INTEGRA 400 which are certified as secondary reference measurement procedures, were considered as reference methods. The CLSI document (EP9-A2) - Method comparison and Bias estimation using patient samples, approved guideline - was used to compare the performance of different HbA1c instruments. The mean of HbA1c in all four types of assays was less than the reference methods (P-value < 0.01).The mean of absolute difference between the reference methods was the least (0.11%). Among the other four tests, Biosystems had the smallest mean of difference (-0.21%), while Pars Azmoon had the highest (-1.18%). Pars Azmoon showed the greatest difference (95% confidence interval) when compared to D10 [-15.5%(-5.7%to -25.3%)] and COBAS INTEGRA [-17% (-9.16% to -24.84%)]. The highest regression slope (B) was found in DS5 method (0.96) in regression model with both reference methods. It can be concluded that although HbA1c standardization programs have resulted in great improvements in the comparability of HbA1c assays, unacceptable errors still exist and further national and international projects are required for standardization of HbA1c measurement. In this situation, it is recommended to use the same laboratory for HbA1c measurement to monitor diabetic patients.

Vitamin D deficiency/insufficiency not only results in disorders of skeletal system, but may also be associated with other nonskeletal diseases. Therefore, measurement of 25-hydroxyvitamin D has received more and more attentions. Common methods for 25-hydroxyvitamin D testing include vitamin D binding protein-based competitive assays, immunoassays, high performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Vitamin D binding protein-based assays and immunoassays can be performed on automated chemistry analyzers, permitting an easy and quick measurement with high throughput. However, significant variability exists among various assays. They cannot separate 25-hydroxyvitamin D3 and D2 either. HPLC method can separate D3 and D2, but the sensitivity and specificity are not as good as mass spectrometry. LC-MS/MS is more specific and sensitive and it can measure D3 and D2 individually. However, the methodology is more complicated and its throughput is relatively low. Also, it requires special instrument and operators who need additional training. In addition, most LC-MS/MS methods are developed and validated by individual laboratories, so the standardization of the method is an urgent problem to be solved. Although the specificity of LC-MS/MS method is high, some metabolites may still interfere with the assay. The reference interval of 25-hydroxyvitamin D is still controversial and how to establish gender- and age-specific reference intervals remains debatable. Besides 25-hydroxyvitamin D, whether its metabolites and free 25-hydroxyvitamin D should be measured is still questionable. Although 25-hydroxyvitamin D testing is primarily used for the diagnosis and treatment of skeletal diseases, epidemiological studies suggest that vitamin D deficiency/insufficiency may be related to the development and prognosis of nonskeletal diseases. However, more evidence is needed in order to determine the causal relationship between them and the mechanism. (Chin J Lab Med, 2017, 40: 744-747) Key words: Vitamin D; Chromatography, liquid; Tandem mass sectrometry

An isocratic reversed phase stability-indicating high-performance liquid chromatographic (HPLC) assay method was developed and validated for quantitative determination of Meclizine HCl in bulk drugs. An isocratic, reversed phase HPLC method was developed to separate the drug from the degradation products, using a Water Nova Pack C18 (250 x 4.6) mm, 5? column and the mobile phase containing 1.0 gm Sodium dihydrogen phosphate and 1.0 gm 1-octaneSulfonic acid salt in 1000ml water filter and mixed. Prepare a homogenous mixture of buffer, methanol and acetonitile. The detection was carried out at wavelength 264 nm. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, selectivity, robustness prove the stability indicating ability of the method.

Part I: Development of a solid phase extraction coupled with high performance liquid chromatography method for the determination of aripiprazole and dehydroaripiprazole in biological fluid Aripiprazole is the first drug with dopamine partial agonist effect for schizophrenia. Dehydroaripiprazole is its major metabolite. The determination and validation of aripiprazole and dehydroaripiprazole in human serum and urine were performed by a combination of solid phase extraction (SPE) and high performance liquid chromatography (HPLC) in this study. The method includes the following steps: 1) pre-treatment of acid-base solutions for deproteination, 2) application of SPE using an Oasis HLB cartridge for cleaning-up and concentration of the samples, 3) HPLC analysis. The recovery of sample pretreatment step was relatively high with recovery rate of 88.20 - 99.83 %. The optimized HPLC conditions were using a C18 X Terra® column, with an isocratic elution consisted of dipotassium phosphate buffer, pH 8.35, and acetonitrile (40 : 60 v/v) at a flow rate of 1.0 mL/min. The concentration of aripiprazole and dehydroaripiprazole could be determined within 5 minutes. The relative standard deviation (RSD) of the peak area for method repeatability (n = 4) and intermediate precision (inter-day, n = 3) were lower than 0.11 % and 5.16 %, respectively. The calibration curves revealed the method that was linear with concentration range between 50 - 1000 ppb for aripiprazole and 50 - 800 ppb for dehydroaripiprazole. Finally, the validated method was successfully applied to analyze serum and urine samples collected from patients receiving the aripiprazole treatment. The developed method can be used to quantitative determination of aripiprazole and dehydroaripiprazole concentration in patients’ serum and urine for therapeutic monitoring and clinical research. Part II: Fingerprint analysis of rhubarb by capillary electrophoresis and ultra-high pressure liquid chromatography This study used capillary electrophoresis (CE) and ultra performance liquid chromatographic (UPLC) method for chromatographic fingerprint analysis of rhubarb. With the application of chemometric approach, chromatographic fingerprint could be used for species differentiation. Ten common constituents in rhubarb, including aloe-emodine, (+)catechin, chrysophanol, emodine, (-)epicatechin gallate, gallic acid, physcion, rhein, sennoside A and sennoside B, were selected for analytical method development. The optimum micellar electrokinetic chromatography (MEKC) conditions were as followed: 30 mM sodium tetraborate / sodium dihydrogen phosphate monohydrate, 30 mM sodium deoxycholate (SDC), pH 8.6 with 26 % acetonitrile (v/v) as background electrolyte. The optimum condition of UPLC method used a Waters Acquity UPLC BEH C18 column for the separation. The mobile phase was composed of 0.05 % phosphate solution (solution A) and acetonitrile (solution B). The gradient profile was ( solution A: solution B): 0 min, 90 : 10; 25 min, 79 : 21; 35 min, 67 : 33; 40 min, 35 : 65; 45, min 35 : 65. The detector wavelength was set at 254 nm for both methods, and the total analytical time was 21 min for CE and 45 min for UPLC. Sixteen samples of Rheum officinale and Rheum tanguticum collected from various sources were analyzed by optimum analytical conditions. Chromatographic fingerprints of CE were subjected to peak alignment and baseline correction for further similarity test. On the other side, analytical results of UPLC show high precision with flat baseline. Chromatographic fingerprints of UPLC were directly used for Principal component analysis (PCA) and similarity test. PCA shows the chromatographic fingerprints of the two species could be successfully classified. The sample showing the least correlation with the representative chromatographic fingerprint was studied for its DNA sequences. DNA analysis demonstrated the sample to be a hybrid rhizome. The developed CE and UPLC chromatographic fingerprint methods could be applied for the quality control of rhubarb.

A sensitive, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the assay of lomefloxacin (LFLX) in raw material and tablet preparations. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was performed on a reversed-phase Phenomenex C18 column (150 x 4.6 mm id, 5 microm particle size) with a mobile phase composed of 1% acetic acid-acetonitrile-methanol (70 + 15 + 15, v/v/v), pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 280 nm. The calibration graph for LFLX was linear from 2.0 to 7.0 mg/mL. The interday and intraday precisions (relative standard deviation) were less than 1.0%. The method was applied for the quality control of commercial LFLX tablets to quantitate the drug.

Cell model for genetic parkinsonism: Can cytological profiling using human olfactory neurosphere derived cells inform the mechanisms of neurodegeneration?

Objective: Objective of the present analytical research work was to develop and validate Spectrophotometric method and Reverse Phase High Performance Liquid Chromatographic method (RP-HPLC Method) for the Eszopiclone bulk and tablets dosage form. Methods: A spectrophotometric method and a RP-HPLC method have been developed and validated for estimation of ESZ in pharmaceutical oral dosage form. Method A (RP-HPLC Method): The RP-HPLC Method for Eszopiclone was developed using Shimadzu HPLC, LC-10, temperature maintained 25 0 C, phenorex Gemini C18 (250 mm 4.60 mm 5?m), as stationary particle, isocratic mode. MeOH: Water (80:20v/v) as mobile phase. Mobile phase was maintained at a flow rate of 1.0 ml/min and detection was carried out at 305 nm. Method B (UV SPECTROMETRY Method): The stock and working standard solutions of the drugs were prepared in methanol. Standard solutions were scanned over the range of 400-200 nm in spectrum mode of spectrophotometer at medium scanning speed using UV spectrophotometer 2450, SHIMADZU. The maximum absorbance for Eszopiclone was found at 305 nm. Both the methods were validated in accordance with ICH guidelines Results: Eszopiclone was found to be linear in the concentration range of 4 - 24 ?g/ml for spectrophotometric method and 5-30 ?g/ml for RP-HPLC method. Retention time was found to be 5.38 min for Eszopiclone. The amount of Eszopiclone in marketed formulation by spectrophotometric method was found to be 100.02 %, the amount of Eszopiclone in marketed formulation by RP-HPLC method was found to be 100.03 %. Interpretation and Conclusion: Results of assay and validation study were found to be satisfactory. So, the methods can be successfully applied for the routine analysis of Eszopiclone.

Objective To compare high performance liquid chromatography (HPLC) and enzyme multiplied irmunoassay technique (EMIT) method in monitoring mycophenolic acid (MPA) in renal transplantation patients.Method Plasma samples were collected from 31 adult kidney transplant recipients at 0.5,1.5,4 and 9 h after administration of mycophenolate mofetil (MMF).All these patients were given MMF,tacrolimus and prednisolone for immunosuppression.Both EMIT and HPLC were employed to determine plasma MPA concentration.Method comparison was performed using Bland & Altman and Passing & Bablok analysis.Result HPLC method and EMIT method exhibited a liner range of 0.1-50.0 mg/L and 0.1-15.0 mg/L.All of MPA concentrations at each time point had no normal distribution.The MPA concentration determined by EMIT was higher than HPLC by 1.44 (-1.88-13.58 rg/L,P＜0.05).The EMIT assay showed a mean positive bias of 29.0％ in concentration,and 19.4％ in AUC0-12h by Bland & Altman plots.The Passing & Bablok regression analysis gave an equation of MPAEMTT =0.3926 + 1.1121 × MPAHPLC,and AUCEMIT =4.7911 + 1.0929 × AUCHPLC.Conclusion It should be considered that the putative therapeutic range for MPA with the EMIT assay is higher than the range using HPLC. Key words: Kidney transplantation; Mycophenolic acid; High performance liquid chromatography; Enzyme multiplied immunoassay technique

A rapid and specific high-performance liquid chromatographic (HPLC) method has been developed for quantitative analysis of famotidine in pharmaceutical dosage forms. The method was applied to medicines on the Turkish market. A LiChrospher RP-18 column, HP-1050 UV-detector and methanol—0·1 M aqueous ammonium acetate (30 : 70) as the solvent system (flow rate 1·0 mL min−1) were used. Quantitative determination of famotidine was carried out at 254 nm wavelength. The method was found to be rapid—under 5 min per assay—and was selective enough to allow the determination of famotidine in the presence of certain excipients.

Development and Validation of a Liquid Chromatographic Method for Concurrent Assay of Weakly Basic Drug Verapamil and Amphoteric Drug Trandolapril inPharmaceutical Formulations

&lt;p&gt;Five Mix Plant Extracts according to different extraction solvents were assessed for its cell viability and anti-inflammatory activity by &lt;em&gt;in vitro&lt;/em&gt; methods. The single plant extract was extracted with 70% ethanol(EtOH) and the mix plants(&lt;em&gt;C.kousa, R.multiflora, T.nucifera, M.basjoo &lt;/em&gt;and&lt;em&gt; S.glabra&lt;/em&gt;) were extracted with EtOH 30%, 70%; Butylene Glycol(BG) 30%, 70%; Propylene Glycol(PG) 30%, 70%; Distilled Water(D.W). Cell viability was measured using the Micro culture tetrazolium (MTT) assay method and Human fibroblast cells, CCD 1102 KERTr were used. The plant extracts with the maximum concentration that none toxic to the cells were evaluated for anti-inflammatory activity. Anti-inflammatory activity was evaluated using lipoxygenase inhibition assay method. A dose response curve was plotted to determine the IC50 values. Results showed that, at the 5 kinds of single plant extracts by 70% EtOH extraction solvent, it showed the IC50 was 280ug/ml of S1, 370ug/ml of S2, 380ug/ml of S3, 170ug/ml of S4 and 190ug/ml of S5. At the mix plant extracts by 7 kinds of extraction solvents (70%, 30% EtOH; 70%, 30% BG; 70%, 30% PG; D.W), it showed the IC50 was 140ug/ml of M E70, 140ug/ml of M E30, 120ug/ml of M BG70, 110ug/ml of M BG30, 120ug/ml of M PG70,&lt;strong&gt; &lt;/strong&gt;136ug/ml of M PG30 and 120ug/ml of M D.W. From the results, it is concluded that when these five plants mixed before extraction, it will extract more active ingredients with anti-inflammatory effects. Further study we will analyzing plants effective single compound using high performance liquid chromatography (HPLC) profiling and progressing the experiments &lt;em&gt;in vivo&lt;/em&gt;.&lt;/p&gt;

Objective To compare the differences of pharmacokinetic parameters of paclitaxel injections and liposome.Methods An high performance liquid chromatography (HPLC) assay method was established to estimate the blood concentrations of paclitaxel after caudal vein administration of 12 mg/kg weigh.The blood concentration-time curves as well as correlated pharmaciokinetic parameters were obtained.Moreover,the data were analyzed with t-test.Results In rats,the mean drug concentration in plasma of paclitaxel injection and paclitaxel liposome were (0.18 ± 0.07)C/(mg · L) and (50 ± 9.2)C/(mg · L),respectively,and there were significant differences between the two formulations (t =5.879,P ＜ 0.01) ;compared with paclitaxel injection,the area under concentration-time curve (AUC) was bigger (17.693 ±2.657 vs 10.812 ± 2.846,t =4.892,P ＜ 0.01),the apparent volume of distribution (Vc) was smaller [(1.629 ±4.91) × 10-3 L vs (5.268 ± 1.121) × 10-3 L,t =3.75,P ＜0.01],the clearance rate(Cl)was slower[(7.2 ± 1.0) × 10-4 L vs (1.2 ±2.7) × 10-4 L,t =4.25,P ＜ 0.01],in the paclitaxel liposome.Conclusions Paclitaxel liposome has higher efficacy,longer release time,higher target tissue concentration,and lower side effects than paclitaxel injection. Key words: Liposomes/pharmacokinetics ; Paclitaxel/pharmacokinetics ; INJECTIO ; Chromatography, high pressure liquid