This article describes the development and validation of a selective, sensitive, and fast high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of atorvastatin and its two metabolites, ortho-hydroxy atorvastatin and para-hydroxy atorvastatin, from human plasma. The analytes were extracted from plasma via solid phase extraction and separated using HPLC with mass spectrometric detection. The method was fully validated and demonstrated good linearity, accuracy, precision, selectivity, sensitivity with a lower limit of quantification of 0.05 ng/mL, and short run time of 3.2 minutes. The method was successfully applied to analyze samples from a clinical pharmac
VALIDATED LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY METHOD FOR DETERMINA...Manik Ghosh
A simple, highly sensitive and rapid LC-MS/MS method has been developed and validated for the quantification of metolazone in rat plasma using irbesartan as internal standard (IS). After simple protein precipitation extraction by acetonitrile, the analyte and IS were extracted from 50 μL plasma sample on an Agilent Poroshell 120, EC- C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column using 5μL injection volume with a total run time of 2 min. Acidified methanol/water mixture was used as a mobile phase. The parent/product ion transitions for metolazone (m/z 366.1/258.9) and IS (m/z 429.2/207.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was found to be linear in the range of 0.05 – 200 metolazone. The method was validated with respect to selectivity, linearity, accuracy, precision, recovery and stability according to accepted regulatory guidelines. The described method was successfully applied to preclinical pharmacokinetic studies of analytes after an oral administration of metolazone (1 mg/kg) in rats.
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
Poster demonstrating the results from the development/verification project for the quantitation of all- trans retinol and alpha tocopherol in human serum.
VALIDATED LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY METHOD FOR DETERMINA...Manik Ghosh
A simple, highly sensitive and rapid LC-MS/MS method has been developed and validated for the quantification of metolazone in rat plasma using irbesartan as internal standard (IS). After simple protein precipitation extraction by acetonitrile, the analyte and IS were extracted from 50 μL plasma sample on an Agilent Poroshell 120, EC- C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column using 5μL injection volume with a total run time of 2 min. Acidified methanol/water mixture was used as a mobile phase. The parent/product ion transitions for metolazone (m/z 366.1/258.9) and IS (m/z 429.2/207.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was found to be linear in the range of 0.05 – 200 metolazone. The method was validated with respect to selectivity, linearity, accuracy, precision, recovery and stability according to accepted regulatory guidelines. The described method was successfully applied to preclinical pharmacokinetic studies of analytes after an oral administration of metolazone (1 mg/kg) in rats.
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
Poster demonstrating the results from the development/verification project for the quantitation of all- trans retinol and alpha tocopherol in human serum.
Synthesis, spectral characterization and bioactivity studies of some S-substi...Jing Zang
A new series of 5-(4-Chlorophenyl)-1,3,4-Oxadiazol-2-thiol derivatives was prepared from 4-chlorobenzoic acid (1) by converting it successively into corresponding ester (2), carbohydrazide (3) and 5-(4-Chlorophenyl)-1,3,4-Oxadiazol-2-thiol (4). Finally the target compounds, 6a-l, were synthesized by stirring 4 with different electrophiles, 5a-l, in DMF using NaH as weak base and activator. The proposed structures of newly synthesized compounds were confirmed by spectroscopic techniques such as 1H-NMR, 13C-NMR, HR-MS and EI-MS. All synthesized compounds were evaluated for their anti-bacterial, antifungal, cytotoxicity and enzyme inhibition activities. The compounds, 6e and 6g exhibited significant inhibition activity against acetyl cholinesterase enzyme (AChE) and 6j moderate activity against butyryl cholinesterase enzyme (BChE). The molecule, 4 exhibited good MIC (minimum inhibitory concentration) value against all the bacterial and fungal strains taken into account.
Stability indicating method development and validation for the simultaneous e...pharmaindexing
Stability indicating method development and validation for the simultaneous estimation of rabeprazole sodium and ketorolac tromethamine in bulk and synthetic mixture by RP-HPLC
Determination of Etodolac in Commercial Formulations by HPLC-UV Methodijtsrd
The aim of this study was to develop and verify a simple, rapid and sensitive high performance liquid chromatography method coupled with UV detector HPLC UV method for the quantitative determination of etodolac in bulk and pharmaceutical dosage forms. Chromatographic separation was performed at ambient conditions on a reverse phase ACE C8 analytical column 250 mm x 4.6 mm ID, 5 umm using the mobile phase containing acetonitrile water 80 20, v v at a flow rate of 1.0 mL min 1. A wavelength of 272 nm was used for etodolok and paracetamol IS . A retention time of 4.21 min and 2.02 min were obtained for etodolac and IS, respectively. The method showed linearity in the range of 0.08 10 µg mL 1 for etodolac R = 0.9999 . The linear regression equations obtained by least square regression method were the ratio of peak area of etodolac and IS =1.559 concentration etodolac µg mL 0.139. The intra day and inter day RE and RSD values of the method were =10.0 and =2.65 , respectively. Limit of detection LOD and limit of quantification LOQ were found to be 0.04 and 0.06 µg mL 1 for etodolac, respectively. A new, simple and sensitive high performance liquid chromatography method was developed and validated for etodolac. The method can be applied for the quantification of etodolac without derivatization in bulk solutions and commercial formulations using the internal standard. Tugrul Cagri Akman | Yucel Kadioglu "Determination of Etodolac in Commercial Formulations by HPLC-UV Method" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29452.pdfPaper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/29452/determination-of-etodolac-in-commercial-formulations-by-hplc-uv-method/tugrul-cagri-akman
chromatography - definition. column chromatography, gas chromatography, high pressure liquid chromatography, thin layer chromatography , paper chromatography - definition and application
Chromatography is a biophysical technique that enables separation, identification and purification of the components of a mixture.
The separation is based on the differential partitioning between the mobile phase and the stationary phase.
Mobile phase – solvent, which move through or over the stationary phase.
Stationary phase – either is solid with high surface area or liquid coated on to a solid; stay still.
Column chromatography is a method for separating mixtures of substances in which a liquid or gaseous solution of mixture (mobile phase) is caused to flow through a tube packed with a finely divided solid (stationary phase ), may be coated with absorbent liquid or through a long capillary tube bearing a thin film of adsorbent liquid.The components of the mixture separates based on the adsorption of the solutes of the solution.
Gas chromatography is technique used to separate and purify individual components from mixture of compounds that can be vaporized. The mobile phase is a gas and the components are separated as vapor. The separation is carried out in a column.
High Performance Liquid Chromatography or high pressure liquid chromatography relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid absorbent material.
Thin layer chromatography is a solid liquid adsorption chromatography. The stationary phase is applied as thin layer on a solid support plate with a liquid mobile phase.
Paper chromatography is analytical method used to separate colored chemicals or substances using a paper as the stationary phase. In this, a thick filter paper comprised the support, and water drops settled in its pores made up the stationary liquid phase. It is a liquid – liquid chromatography.
Development and validation of rphplc method for nsai ds in combined pharmaceu...IJSIT Editor
The present research work includes the development and validation of High performance liquid
chromatography method for simultaneous estimation of Etirocoxib (ETX )and Paracetamol (PCM) which are
very much of useful in multiple therapies rather than the use of single drug formulation, because of multiple
actions, fever side effect ,and quicker relief. We aimed to design a quick and simple method, suitable for the
determination of identity, strength. The UV spectroscopic method were developed on standard ETX and PCM
using methanol as standard solution and also, the standard stock solution of both ETX and PCM were diluted
with 2%hydrochloric acid to obtain concentration 2.0µg/ml and 16.6µg/ml.The spectrum was recorded in the
range of 400-200nm.The RP- HPLC method were developed on standard ETX and PCM which performed on C-
18 column using mobile phase composed by acetonitrile :water 50:50(v/v) at a flow rate of 1 ml/min, with
injection volume 20µl and Ph 6.8 can be adjusted using orthophosphoric acid.
N,N-Diallyl-5-methoxytryptamine (also known as 5-MeO-DALT or colloquially as Foxtrot) is a lesser-known psychedelic substance of the tryptamine class that produces short-lived psychedelic effects when administered. It is structurally related to tryptamines like 5-MeO-DiPT and DALT, although it is reported to produce distinct effects.
https://brcshops.com/product/5-meo-dalt/
The Effect of Atorvastatin (Lipitor) on the Duration of Survival of Allogenei...Nabil Zeidan
Aim: To evaluate the immunomodulatory effect of using non-cholesterol lowering dose
of atorvastatin (AS) on skin allograft survival and on tumor growth in mice.
Study Design: Experimental Study.
Place and Duration of Study: Department of Experimental Pathology, Immunology and
Microbiology, Faculty of Medicine, American University of Beirut; 2011-2012.
Methodology: BALB/c mice were transplanted with skin allografts from C57BL/6 mice
and given either AS alone or in combination with immunosuppressive agents. Average
survival days of skin allografts were recorded and serum levels of interleukin-1β (IL-1β)
and interferon-γ (IFN-γ) were quantified. BALB/c mice and C57BL/6 mice were
challenged intraperitoneally with B16F10 melanoma cancer cells (cancer cell line
syngeneic to C57BL/6 mice) and were then treated with AS. They were observed regularly for tumor growth.
Results: The results indicated that in transplant mice AS given alone or in combination
with immunosuppressive agents prolonged allograft survival time through noncholesterol
lowering mechanisms in spite of a non-significant change in serum cytokine
levels. Furthermore, AS treatment enhanced tumor growth in C57BL/6 mice and
promoted tumor growth in BALB/C mice.
Conclusion: It can be speculated that AS down expresses TLR and modifies MHC
presentation resulting in hindering the generation of an innate and adaptive immune
response.
Synthesis, spectral characterization and bioactivity studies of some S-substi...Jing Zang
A new series of 5-(4-Chlorophenyl)-1,3,4-Oxadiazol-2-thiol derivatives was prepared from 4-chlorobenzoic acid (1) by converting it successively into corresponding ester (2), carbohydrazide (3) and 5-(4-Chlorophenyl)-1,3,4-Oxadiazol-2-thiol (4). Finally the target compounds, 6a-l, were synthesized by stirring 4 with different electrophiles, 5a-l, in DMF using NaH as weak base and activator. The proposed structures of newly synthesized compounds were confirmed by spectroscopic techniques such as 1H-NMR, 13C-NMR, HR-MS and EI-MS. All synthesized compounds were evaluated for their anti-bacterial, antifungal, cytotoxicity and enzyme inhibition activities. The compounds, 6e and 6g exhibited significant inhibition activity against acetyl cholinesterase enzyme (AChE) and 6j moderate activity against butyryl cholinesterase enzyme (BChE). The molecule, 4 exhibited good MIC (minimum inhibitory concentration) value against all the bacterial and fungal strains taken into account.
Stability indicating method development and validation for the simultaneous e...pharmaindexing
Stability indicating method development and validation for the simultaneous estimation of rabeprazole sodium and ketorolac tromethamine in bulk and synthetic mixture by RP-HPLC
Determination of Etodolac in Commercial Formulations by HPLC-UV Methodijtsrd
The aim of this study was to develop and verify a simple, rapid and sensitive high performance liquid chromatography method coupled with UV detector HPLC UV method for the quantitative determination of etodolac in bulk and pharmaceutical dosage forms. Chromatographic separation was performed at ambient conditions on a reverse phase ACE C8 analytical column 250 mm x 4.6 mm ID, 5 umm using the mobile phase containing acetonitrile water 80 20, v v at a flow rate of 1.0 mL min 1. A wavelength of 272 nm was used for etodolok and paracetamol IS . A retention time of 4.21 min and 2.02 min were obtained for etodolac and IS, respectively. The method showed linearity in the range of 0.08 10 µg mL 1 for etodolac R = 0.9999 . The linear regression equations obtained by least square regression method were the ratio of peak area of etodolac and IS =1.559 concentration etodolac µg mL 0.139. The intra day and inter day RE and RSD values of the method were =10.0 and =2.65 , respectively. Limit of detection LOD and limit of quantification LOQ were found to be 0.04 and 0.06 µg mL 1 for etodolac, respectively. A new, simple and sensitive high performance liquid chromatography method was developed and validated for etodolac. The method can be applied for the quantification of etodolac without derivatization in bulk solutions and commercial formulations using the internal standard. Tugrul Cagri Akman | Yucel Kadioglu "Determination of Etodolac in Commercial Formulations by HPLC-UV Method" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29452.pdfPaper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/29452/determination-of-etodolac-in-commercial-formulations-by-hplc-uv-method/tugrul-cagri-akman
chromatography - definition. column chromatography, gas chromatography, high pressure liquid chromatography, thin layer chromatography , paper chromatography - definition and application
Chromatography is a biophysical technique that enables separation, identification and purification of the components of a mixture.
The separation is based on the differential partitioning between the mobile phase and the stationary phase.
Mobile phase – solvent, which move through or over the stationary phase.
Stationary phase – either is solid with high surface area or liquid coated on to a solid; stay still.
Column chromatography is a method for separating mixtures of substances in which a liquid or gaseous solution of mixture (mobile phase) is caused to flow through a tube packed with a finely divided solid (stationary phase ), may be coated with absorbent liquid or through a long capillary tube bearing a thin film of adsorbent liquid.The components of the mixture separates based on the adsorption of the solutes of the solution.
Gas chromatography is technique used to separate and purify individual components from mixture of compounds that can be vaporized. The mobile phase is a gas and the components are separated as vapor. The separation is carried out in a column.
High Performance Liquid Chromatography or high pressure liquid chromatography relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid absorbent material.
Thin layer chromatography is a solid liquid adsorption chromatography. The stationary phase is applied as thin layer on a solid support plate with a liquid mobile phase.
Paper chromatography is analytical method used to separate colored chemicals or substances using a paper as the stationary phase. In this, a thick filter paper comprised the support, and water drops settled in its pores made up the stationary liquid phase. It is a liquid – liquid chromatography.
Development and validation of rphplc method for nsai ds in combined pharmaceu...IJSIT Editor
The present research work includes the development and validation of High performance liquid
chromatography method for simultaneous estimation of Etirocoxib (ETX )and Paracetamol (PCM) which are
very much of useful in multiple therapies rather than the use of single drug formulation, because of multiple
actions, fever side effect ,and quicker relief. We aimed to design a quick and simple method, suitable for the
determination of identity, strength. The UV spectroscopic method were developed on standard ETX and PCM
using methanol as standard solution and also, the standard stock solution of both ETX and PCM were diluted
with 2%hydrochloric acid to obtain concentration 2.0µg/ml and 16.6µg/ml.The spectrum was recorded in the
range of 400-200nm.The RP- HPLC method were developed on standard ETX and PCM which performed on C-
18 column using mobile phase composed by acetonitrile :water 50:50(v/v) at a flow rate of 1 ml/min, with
injection volume 20µl and Ph 6.8 can be adjusted using orthophosphoric acid.
N,N-Diallyl-5-methoxytryptamine (also known as 5-MeO-DALT or colloquially as Foxtrot) is a lesser-known psychedelic substance of the tryptamine class that produces short-lived psychedelic effects when administered. It is structurally related to tryptamines like 5-MeO-DiPT and DALT, although it is reported to produce distinct effects.
https://brcshops.com/product/5-meo-dalt/
The Effect of Atorvastatin (Lipitor) on the Duration of Survival of Allogenei...Nabil Zeidan
Aim: To evaluate the immunomodulatory effect of using non-cholesterol lowering dose
of atorvastatin (AS) on skin allograft survival and on tumor growth in mice.
Study Design: Experimental Study.
Place and Duration of Study: Department of Experimental Pathology, Immunology and
Microbiology, Faculty of Medicine, American University of Beirut; 2011-2012.
Methodology: BALB/c mice were transplanted with skin allografts from C57BL/6 mice
and given either AS alone or in combination with immunosuppressive agents. Average
survival days of skin allografts were recorded and serum levels of interleukin-1β (IL-1β)
and interferon-γ (IFN-γ) were quantified. BALB/c mice and C57BL/6 mice were
challenged intraperitoneally with B16F10 melanoma cancer cells (cancer cell line
syngeneic to C57BL/6 mice) and were then treated with AS. They were observed regularly for tumor growth.
Results: The results indicated that in transplant mice AS given alone or in combination
with immunosuppressive agents prolonged allograft survival time through noncholesterol
lowering mechanisms in spite of a non-significant change in serum cytokine
levels. Furthermore, AS treatment enhanced tumor growth in C57BL/6 mice and
promoted tumor growth in BALB/C mice.
Conclusion: It can be speculated that AS down expresses TLR and modifies MHC
presentation resulting in hindering the generation of an innate and adaptive immune
response.
Veeprho Pharmaceuticals s.r.o is a supplier of Atorvastatin Impurities like Trans Atorvastatin Impurity, Atorvastatin Lactone Impurity and Atorvastatin Related compound A, B,C, D, E etc. to pharmaceuticals industry.
Atorvastatin: Statins in CVD management. Is just lipid lowering enough Dr Vivek Baliga
When it comes to management of cardiovascular diseases, are achieving lipid lowering targets sufficient. Here Dr Vivek Baliga, Consultant Internal medicine discusses the additional benefits of statins in CVD in India.
Dyslipidemia management an evidence based approachDr Vivek Baliga
In this presentation by Dr Vivek Baliga, he discusses the different available statins and how you can choose the right one in different clinical situations. See articles from Dr Baliga on http://drvivekbaliga.net
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Structural elucidation, Identification, quantization of process related impur...IOSR Journals
Major process related unknown impurity associated with the synthesis of Hydralazine hydrochloride bulk drug was detected by high performance liquid chromatography (HPLC) and was subjected to high resolution accurate liquid chromatography mass spectroscopy (HR/AM-LCMS) for identification. The proposed impurity was isolated from Hydralazine hydrochloride active pharmaceutical ingredient (API) by preparative chromatographic method and was injected on HPLC for comparison of retention time with that of the unknown process related impurity in Hydralazine hydrochloride. The molecular ion peak of preparatively isolated impurity and that of unknown process related impurity in Hydralazine hydrochloride were compared for confirmation. The postulated structure was unambiguously confirmed with the help of HR/AM- LC MS/MS, NMR and FTIR data proposed to be 1-(2-phthalazin-1-ylhydrazino)phthalazine (Hazh Dimer). This impurity of Hydralazine hydrochloride is not been previously reported. A rapid Acquity H-class gradient method with runtime of 15.0min was developed for Quantitation on Unisphere Cyno column and validated for parameters such as accuracy, precision, linearity and range, robustness. The LOD and LOQ of method were 0081% and 0.0246% respectively.
Analytical Method Development and Validation for the Estimation of Zolmitript...ijtsrd
In this work the authors have proposed a simple, specific, economic and accurate reverse phase liquid chromatographic method for the estimation of Zolmitriptan as an active pharmaceutical ingredient and in pharmaceutical formulation. The main objective of the current research paper is to To develop simple, precise and accurate RP HPLC method for Zolmitriptan also to validate the developed method as per ICH guideline Q2R1 and to explore the applicability of the method in finished product formulation for estimation of Zolmitriptan during its lifecycle. The objective was achieved by optimized condition with Phonemenex C18 column 150mm×4.6mm , 5µm. And mobile phase Phosphate buffer pH 3.5 85 Methanol 15. The separation was done with a flow rate of 0.9ml min, detection with 224nm. The retention was found to be 3.57 minute. LOD and LOQ were found to be 2.45 and 7.42 respectively. So in order to obtain the correct results various validations methods are performed to get the results. The results obtained from those validation methods are plotted in the form of the charts as well as the different curves. Mr. Rahul M. Sagde | Mr. Pawan N. Karwa | Mr. Vivek M. Thorat | Sanjay S. Jadhav "Analytical Method Development and Validation for the Estimation of Zolmitriptan by RP HPLC Method" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-5 , August 2019, URL: https://www.ijtsrd.com/papers/ijtsrd26474.pdfPaper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/26474/analytical-method-development-and-validation-for-the-estimation-of-zolmitriptan-by-rp-hplc-method/mr-rahul-m-sagde
A REVIEW ON ETORICOXIB AND PREGABALIN IN METHOD VALIDATION BY RP-HPLCUpexaBavadiya
Development and Validation for Simultaneous Estimation of Etoricoxib and Pregabalin in Bulk and Tablet Dosage Form by RP-HPLC 2K20,GTU,MASTER IN PHARMACY IN QA
Development and validation of hplc method for determination of theophylline a...IJSIT Editor
A stable, simple, rapid, precise, accurate HPLC method for analysis of Theophyllinee and 1-Methyl
Uric Acid was developed and validated as per ICH guidelines without need of any internal standard.
Separation was carried out using X’terra RP18 (250*4.6) mm, 5µ column with potassium dihydrogen
orthophosphate buffer (pH 3): acetonitrile (30:70 v/v) as mobile phase with flow rate 1 mL min-1. The
parameters studied were retention time, linearity and range, accuracy, precision. The proposed method can
be used for determination of Theophylline and 1-Methyl Uric Acid from Human plasma.
ETORICOXIB AND PREGABALIN OF METHOD DEVLOPMENT IN RPHPLC BY UPEXA BAVADIYAUpexaBavadiya
Development and Validation for Simultaneous Estimation of Etoricoxib and Pregabalin in Bulk and Tablet Dosage Form by RP-HPLC 2K21 GTU MASTER IN PHARMACY BY UPEXA BAVADIYA
Root cause Analysis (RCA) & Corrective and Preventive action (CAPA) in MRCT d...Bhaswat Chakraborty
This presentation describes Identification & differentiation of Protocol deviation & violation; Different methods of RCA & best suitable method for Multiregional Clinical Trial; CAPA management and CAPA application to other trial sites/CRO/SMO/ Country that is involved in same trial (Strategic Management and application of CAPA in MRCT)
This presentation gives effective solutions to outliers issue in bioequivalence trials. It described what would be acceptable to Regulatory agencies as well as some new approaches.
Equivalence approches for complex generics DIA 11 april 2019 Bhaswat Chakraborty
This is a workshop that i gave a few days ago on bioequivalence of complex generics like peptides, polymers, liposomes, colloids, ophthamic and topical produtcts.
Clinical trials that are needed for efficacy & safety evidence of Medical devices include feasibility (pilot) and Pivotal trials. An extended battery of preclinical trials are also needed for high risk devices.
Writing Science papers for for publication requires something more thatn creativity. Target journals, content organization, wrting style, elegance and referencing are equally important.
Multidisc review of NDAs and BLAs nipicon 2018 Dr. ChakrabortyBhaswat Chakraborty
NDAS and BLAs cannot be authoritatively reviewed these days until experts from different disciplines act together like a team. This presentation give some foundational points and an illustrative example in that regard.
Teaching by stories, anecdotes and historical facts sept 25 2018Bhaswat Chakraborty
Many difficult principles in science and humanities can be taught best by a story (of its discovery), by an anecdote or some historical facts about them.
Orientation and Adaptation for Post-Graduate Pharmacy ProgramsBhaswat Chakraborty
PG Pharmacy programs are more focused and professionally oriented than the undergraduate counterpart. Many soft skills are required along with the curricular competence for excellence at the PG level.
Scientific integrity calls for some basic originality. Plagiarism can destroy this original creativity and ideation. This presentation defines plagiarism (stealing from others' works) and some of the creative and systematic remedies.
Best Practices to Risk Based Data Integrity at Data Integrity Conference, Lon...Bhaswat Chakraborty
Data integrity can be implemented using several approaches. One of the most effective ways to implement DI is a risk based approach. The speaker elaborates this.
There are several dimensions in Pharmaceutical ethics -- Practice-, research- and community oriented. This presentation mainly deals with Clinical research oriented Ethics.
Young pharmaceutical scientists are and can get involved in all aspects of new drug discovery and development. They have to be appropriately qualified, trained and experienced though,
This presentation mainly deals with clinical development of biosimilar products. It also gives enough on non-clinical development so that the audience is well oriented.
High variability in PK can be a characteristic of certain drug products which require different from ordinary strategies and study designs for establishing bioequivalence.
High variability in PK can be a characteristic of certain drug products which require different from ordinary strategies and study designs for establishing bioequivalence.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Sudheer Mechineni, Head of Application Frameworks, Standard Chartered Bank
Discover how Standard Chartered Bank harnessed the power of Neo4j to transform complex data access challenges into a dynamic, scalable graph database solution. This keynote will cover their journey from initial adoption to deploying a fully automated, enterprise-grade causal cluster, highlighting key strategies for modelling organisational changes and ensuring robust disaster recovery. Learn how these innovations have not only enhanced Standard Chartered Bank’s data infrastructure but also positioned them as pioneers in the banking sector’s adoption of graph technology.
Dr. Sean Tan, Head of Data Science, Changi Airport Group
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Atorvastatin dta
1. Research Article
Drug Testing
and Analysis
Received: 16 June 2010 Revised: 2 October 2010 Accepted: 13 October 2010 Published online in Wiley Online Library:
(www.drugtestinganalysis.com) DOI 10.1002/dta.228
Simultaneous estimation of atorvastatin
and its two metabolites from human plasma
by ESI-LC-MS/MS
Chinmoy Ghosh,∗
Ina Jain, Shashank Gaur, Niraj Patel, Anita Upadhyay
and Dr. Bhaswat S. Chakraborty
A selective, sensitive, and fast high performance liquid chromatography (HPLC) method with mass spectrometric (MS) detection
mode has been developed and validated completely in human plasma. Atorvastatin (ATO), p-hydroxy atorvastatin (p-HATO),
o-hydroxy atorvastatin (o-HATO) and internal standard (IS) are extracted from human plasma via solid phase extraction (SPE)
technique. After elution, the solution is evaporated, then reconstituted with 250 µL of Mobile Phase and analyzed using
HPLC/MS/MS system. An isocratic mode is used to separate interference peaks using a Symmetry C-18, 75 × 4.6 mm ID, 3.5 µ,
column. The m/z of ATO, o-HATO and p-HATO are 559.2/440.2, 575.3/440.4 and 575.0/440.4 respectively. Linearity ranges are
0.05 to 252.92 ng/mL for ATO, p-HATO and o-HATO respectively. Calibration functions, lower limit of quantitation (LLOQ),
stability, intra- and inter-day reproducibility, accuracy, and recovery are estimated. This method is free from matrix effects
and any abnormal ionization. This method was successfully applied to a single dose 80 mg tablet bioequivalence (BE) study of
Atorvastatin. Copyright c 2011 John Wiley & Sons, Ltd.
Keywords: atorvastatin; p-hydroxy atorvastatin; o-hydroxy atorvastatin; solid phase extraction; matrix effects; positional isomer
Introduction
Atorvastatin (ATO; Figure 1) a member of the drug class known
as statins, is used for lowering blood cholesterol. ATO inhibits 3-
hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the
rate-determining enzyme located in hepatic tissue that produces
mevalonate, a small molecule used in the synthesis of cholesterol
and other mevalonate derivatives. This lowers the amount of
cholesterol produced which in turn lowers the total amount of
low-density lipoprotein (LDL) cholesterol. The most important
abnormalities in the lipid profile are an increase in triglyceride
levels, the presence of small, dense LDL particles and low
high-density lipoprotein (HDL) cholesterol levels. The increase
in triglyceride levels is due to elevated levels of very-low-density
lipoprotein (VLDL) remnants and intermediate-density lipoprotein
(IDL). Each of these parameters has been associated with increased
risk of cardiovascular disease.[1] ATO has developed a well-defined
role in the primary and secondary prevention of cerebrovascular
disease, and appears to have a particularly prominent place in
preventing such disease in coronary heart disease (CHD) patients,
and in the post-stroke and post-transient ischemic attack (TIA)
setting in patients without CHD.[2] Statin therapy also decreases
platelet activation and aggregation, and thereby may decrease
the propensity toward thrombosis. Statin therapy inhibits tissue
factor expression by macrophages, which plays an integral role
in blood coagulation and is an important determinant of plaque
thrombogenicity. There also may be a reduction in plasminogen
activator inhibitor activity, which would facilitate fibrinolysis. All of
thesefactorsmaycontributetowardsdecreasedriskforstrokeseen
in the statin studies. These statin effects may lead to decreased
thrombus formation and thereby influence the development of
clinical activity related to atherosclerotic plaque.
Several HPLC [3,4] and LC-MS/MS [5–18] methods have been
reported for estimation of ATO alone or with its metabolites,
i.e. ortho hydroxy atorvastatin (o-HATO) and para hydroxy
atorvastatin(p-HATO).Amongallthesemethods,fewofthemhave
LLOQvaluesaslowas0.10 ng/mLandothersaremuchhigher.[3–18]
Those methods are either compromised by long analysis times or
required the use of more sensitive instrumentation to achieve
this sensitivity. However, there were no reported methods with
LLOQ value as low as 0.05 ng/mL for ATO alone or in combination
with its metabolite. This low LLOQ value will help to get better
time-concentration profile of the analyte(s). This low LLOQ value
also helps to apply the same analytical method for lower dosage
of ATO. Moreover, some methods had used either one or two
stable isotope labelled ATO, as an internal standard,[12,16] which
is a very expensive material. But in this reported method only
enalapril was used as IS, which is easily available. Moreover, some
methods have long analysis time,[3,6,8,9,13]
where as this reported
method has very short analysis time of 3.2 min. So this method
possesses the highest sensitivity with very short analysis time and
good resolution between the positional isomers, though it is very
difficult to achieve such a good resolution between the positional
isomers with short analysis time.
∗ Correspondence to: Chinmoy Ghosh, Research Scientist, Bio-analytical
Department, Contract Research Organization, Cadila Pharmaceuticals Lim-
ited1389, Trasad road, Dholka-387 810, Dist – Ahmedabad, Gujarat, India.
E-mail: chinmoy ghosh@yahoo.com
Bio-analyticalDepartment,CadilaPharmaceuticalsLimited,1389-Trasadroad,
Dholka, Gujarat, India
Drug Test. Analysis (2011) Copyright c 2011 John Wiley & Sons, Ltd.
2. Drug Testing
and Analysis C. Ghosh et al.
Figure 1. Chemical structure of A) Atorvastatin, B) o-Hydroxy Atorvastatin
and C) p-Hydroxy Atorvastatin.
Experiment
Apparatus and software
The HPLC system with an auto sampler was a Shimadzu LC-20AD
(Shimadzu, Japan) coupled with Applied Biosystem Sciex (MDS
Sciex ON, Canada) API 4000 Tandem mass spectrometer. The auto
sampler was SIL-HTC from Shimadzu, Japan. The solvent delivery
modulewasLC-20ADfromShimadzu,Japan.Thechromatographic
integration was performed by Analyst software (version: 1.4.2;
Applied Biosystems). Positive pressure unit used for SPE was
from Orochem technologies Inc (Lombard, IL, USA). The Caliper
turbovap LV concentration workstation used to evaporate the
samples was purchased from Caliper Life Sciences (Hopkinton,
MA, USA).
Chemicals and reagents
ATOcalciumwasprocuredfromCadilaPharmaceuticalLtd,Dholka,
Ahmedabad, India; p-Hydroxy atorvastatin calcium salt and o-
Hydroxy atorvastatin calcium salt were purchased from VARDA
Biotech Ltd, Mumbai, India. Acetic acid and formic acid was
procured from Merck Specialities Pvt. Ltd, Mumbai, India. Water
used was collected from water purification systems (Billerica,
Massachusetts Milli Q, Milli Pore, USA) installed in laboratory.
Methanol and acetonitrile were of HPLC grade and were supplied
by J. T. Baker (Billerica, Massachusetts, USA. Fresh frozen human
Table 1. Tandem mass spectrometer main working parameters
Parameter Value
Source temperature, ◦
C 400
Dwell time per transition, msec 500
Ion source gas 1, psi 30
Ion source gas 2, psi 50
Curtain gas, psi 10
Collision gas, psi 11
Ion spray voltage, V 4600
Entrance potential, V
10(atorvastatin)
11 (ortho-hydroxyatorvastatin)
11 (para-hydroxyatorvastatin),
11 (IS)
Declustering potential, V
60 (atorvastatin)
120 (ortho-hydroxyatorvastatin)
130 (para-hydroxyatorvastatin),
100 (IS)
Collision energy, V
32 (atorvastatin)
30 (ortho-hydroxyatorvastatin)
30 (para-hydroxyatorvastatin)
25 (IS)
Collision cell exit potential, V
12 (atorvastatin)
12 (ortho-hydroxyatorvastatin)
10 (para-hydroxyatorvastatin)
12 (IS)
Resolution Unit
Mode of analysis Positive
Ion transition for atorvastatin, m/z 559.2/440.2
ortho-hydroxyatorvastatin, m/z 575.3/440.4
para-hydroxyatorvastatin, m/z 575.0/440.4
IS (enalapril), m/z 377.1/234.2
plasma(K2-EDTAasanticoagulant)wasusedduringvalidation,and
was supplied by Clinical department of Cadila Pharmaceuticals
Limited, 1389-Trasad road, Dholka, Gujarat, India. Plasma was
stored into −70 ± 5 ◦
C.
Standards and working solutions
Individual stock standard solution of ATO, p-HATO and o-
HATO containing 1 mg/mL was prepared by dissolving pure
compound in methanol. Intermediate and working solu-
tions of ATO, p-HATO & o-HATO were prepared from cor-
responding stock solutions by diluting with diluent (wa-
ter: methanol 50 : 50 v/v). Calibration standards were es-
tablished between 0.05 to 252.92 ng/mL of ATO, p-HATO
and o-HATO, using eight concentration levels. Quality con-
trol (QC) standards of three different levels low (LQC)
(0.15 ng/mL), medium (MQC) (26.55 ng/mL) and high (HQC)
(140.35 ng/mL) for ATO, p-HATO and o-HATO were also pre-
pared. All these stock solutions, calibration standards and
QC samples were stored at 4 ± 2 ◦
C. These solutions were
found to be stable and used for the complete method
validation.
www.drugtestinganalysis.com Copyright c 2011 John Wiley & Sons, Ltd. Drug Test. Analysis (2011)
3. Determination of atorvastatin and its two positional isomers
Drug Testing
and Analysis
Figure 2. Representative chromatogram of Plasma Blank of A) o-HATO, B) p-HATO, C) ATO and D) IS.
Chromatographic conditions
Chromatographic separation was performed on a Waters Symme-
try C18, 75×4.6 mm ID, 3.5 µ analytical column. The mobile phase
used was a mixture of acetonitrile and 0.60% (v/v) acetic acid in
water at a ratio of 70 : 30 v/v. The flow rate was 0.500 mL/min.
Total analysis time of single injection is 3.20 min. Column oven
temperature was set to 40 ◦
C.
Mass spectrometric conditions
The mass spectrometric parameters to determine the ATO,
o-HATO, p-HATO and IS are presented in Table 1.
Sample treatment
Solid phase extraction technique was used to extract the ATO,
p-HATO and o-HATO from human plasma samples. 2% v/v formic
acidsolutionwasusedforprovidingacidicmedium.500 µLplasma
samples were transferred to RIA vials for analysis. 50 µL of IS
(250 ng/mL) sample was added and the samples were vortexed
for 15 s, followed by the addition of 100 µL of 2% formic acid
solution (v/v). The samples were again vortexed for 15 s. 500 µL
of Milli-Q water was added to dilute the plasma sample and
vortexed for 15 s. Oasis HLB 1 cc 30 mg SPE cartridge (Milford
Waters Corporation, USA) was conditioned with 1 mL methanol,
followed by equilibration with 1 mL of Milli-Q Water. Then the
sample was loaded and washed with 1 mL of Milli-Q Water. Then
the cartridge was dried under nitrogen for 1 min. and the sample
was eluted with 1 mL of methanol, followed by evaporation under
the nitrogen stream at 40 ◦
C. The dry residue was reconstituted
with 250 µL of mobile phase.
Results and Discussions:
Optimization of chromatographic condition and sample
clean-up:
The successful analysis of the analyte in biological fluids using LC-
MS/MS relies on the optimization of chromatographic conditions,
sample preparation techniques, chromatographic separation and
post column detection, etc.[19,20] Thus, for better selectivity and
sensitivity, different types of columns and mobile phases were
used. The length of the column varied from 50 mm to 150 mm,
and the particle size varied from 3.5 µ to 5 µ. Columns of
Drug Test. Analysis (2011) Copyright c 2011 John Wiley & Sons, Ltd. www.drugtestinganalysis.com
4. Drug Testing
and Analysis C. Ghosh et al.
Figure 2. (Continued).
different stationary phases like C8, C18, cyano etc., were used
which demonstrated significant effects on interference, resolution
between positional isomers and peak shape. There was almost
no resolution was obtained with a cyano column, whereas the
use of C8 resulted in poor resolution. Finally, a Waters Symmetry
C18, 75 × 4.6 mm ID, analytical column of 3.5 µ particle size was
selected for analysis.
The influences of strength of the buffer, pH and types of organic
modifier on the signal intensities were also studied. Based on the
peak intensity of the ATO, p-HATO, o-HATO and IS, 0.6% acetic
acid (v/v) and acetonitrile (30 : 70 v/v) as mobile phase at a flow
rate of 0.500 mL/min were selected for further studies. Initially
90% acetonitrile: 10% of 0.6% acetic acid (v/v) at a flow rate
of 0.500 mL/min was tried. However, a very high proportion of
organic phase led to improper elution and peak deformation.
Therefore, the 70 : 30 (v/v) organic phase to buffer were selected
as optimum.
Different extraction techniques were tried to extract ATO and
its two metabolites from plasma samples. In protein precipitation,
almost no recovery was obtained even in different conditions like
acidic, basic or neutral. Then liquid-liquid extraction was carried
out,resultinginlowrecovery,whichwasnotsufficienttodetectthe
LLOQvalue.Finally,solidphaseextractiontechniquewasadopted.
In this method different cartridges were tested, but except HLB
1 mg 30 cc cartridge, other cartridges showed very poor and
inconsistent recovery. So to get the optimum and consistent
recovery for all three analytes, the above mentioned extraction
technique was finalized for further study sample analysis.
Method validation
Afullvalidationaccording totheFDAguidelines[21] wasperformed
for the assay in human plasma.
Aqueous solution linearity
Aqueous solution linearity of calibration standards, i.e. spiking so-
lution checking was assessed by subjecting the spiked concentra-
tions and the respective peak areas using 1/X2 (X – concentration)
www.drugtestinganalysis.com Copyright c 2011 John Wiley & Sons, Ltd. Drug Test. Analysis (2011)
5. Determination of atorvastatin and its two positional isomers
Drug Testing
and Analysis
Figure 3. Representative chromatogram of LLOQ (0.05 ng/mL) of A) o-HATO, B) p-HATO, C) ATO and D) IS.
linear least-squares regression analysis. The calibration curves had
a correlation coefficient (r) of 0.9900 or better. In aqueous solu-
tion linearity test all calibration standards accuracy were within
85–115%, except LLOQ where it was 80–120%.
Specificity and selectivity
Six different lots of plasma along with one lipemic plasma and one
haemolyzed plasma were analyzed to ensure that no endogenous
interferences were present at the retention time of ATO, p-HATO,
o-HATO and IS. Six LLOQ level samples along with plasma blank
from the respective plasma lots were prepared and analyzed. In
all plasma blanks, the response at the retention time of ATO,
p-HATO and o-HATO were less than 20% of LLOQ response and at
the retention time of IS, the response was less than 5% of mean
IS response in LLOQ. Figure 2 shows a typical chromatogram of
plasma blank; Figure 3 represents the chromatogram of LLOQ.
Accuracy and precision
For the validation of the assay, QC samples were prepared at three
concentration levels (low, medium, and high). The respective
concentrations were 0.15, 26.55, and 140.35 ng/mL for ATO, p-
HATO and o-HATO. Six replicates of each QC sample were analyzed
together with a set of calibration standards. The accuracy of
each plasma sample preparation was determined by injection
of calibration samples and LQC, MQC, and HQC samples in six
replicates on each day for three days. The obtained accuracy and
precision (inter- and intra-day) data are presented in Table 2 for
ATO, p-HATO and o-HATO. The result showed that the analytical
method was accurate, as the accuracy was within the acceptance
limits of 100 ± 15% at their respective concentration levels. The
precision around the mean value was never greater than 15%
at any of the concentrations studied. Figure 4 represents the
chromatogram of upper limit of quantization (ULOQ).
Recovery study
Recovery was evaluated by comparing extracted QC samples of
three different levels in six replicate with aqueous samples of same
level. The mean recovery of ATO was 66.18% and the %CV of mean
recovery of all its three QC levels was10.28, where as the mean
recovery of p-HATO was 54.01 and the %CV of mean recovery of
Drug Test. Analysis (2011) Copyright c 2011 John Wiley & Sons, Ltd. www.drugtestinganalysis.com
6. Drug Testing
and Analysis C. Ghosh et al.
Figure 3. (Continued).
all its three QC levels was 7.87 and the mean recovery of o-HATO
was 45.36% and the %CV of mean recovery of all its three QC levels
was 10.36. Recovery of internal standard was 44.45%.
Haemolysis effects
To determine the haemolysis effects, six haemolysed plasma
blanks and QC samples at two different concentration levels,
i.e. LQC (0.15 ng/mL) and HQC (140.35 ng/mL) were prepared.
Six replicates of each QC sample were analyzed together with
a set of calibration standards prepared in normal plasma. The
accuracy of each sample preparation was determined by injection
of calibration samples and two QC samples in six replicates. The
average % accuracy of LQC level was 99.83 and for HQC level was
98.67 for ATO; % accuracy for LQC level was 103.00 and for HQC
level was 104.46 for p-HATO; and % accuracy for LQC level was
103.36 and for HQC level was 101.93 for o-HATO. The %CV of LQC
was 4.29 and for HQC was 2.63 for ATO; %CV of LQC was 5.31and
for HQC was 3.30 for p-HATO; and %CV of LQC was 4.09 and for
HQC was 1.93 for o-HATO.
Matrix effects
The effect of human plasma constituents over the ionization of
ATO, p-HATO, o-HATO and IS were determined by comparing the
responses of the post-extracted plasma standard QC samples (n =
18) with the response of analytes from aqueous standard samples
at low, medium, and high QC of equivalent concentrations. The
% accuracy for ATO for LQC was 97.51 and for HQC was 98.70; %
CV LQC and HQC was 4.22 and 0.96, respectively; for p-HATO %
accuracy for LQC was 94.42 and for HQC was 100.19; % CV of LQC
and HQC was 4.48 and 1.12, respectively; for o-HATO % accuracy
for LQC was 98.90 and for HQC was 101.12; % CV of LQC and HQC
was 4.10 and 1.32, respectively.
www.drugtestinganalysis.com Copyright c 2011 John Wiley & Sons, Ltd. Drug Test. Analysis (2011)
7. Determination of atorvastatin and its two positional isomers
Drug Testing
and Analysis
Figure 4. Representative chromatogram of ULOQ (252.92 ng/mL) of A) o-OATO, B) p-HATO, C) ATO and D) IS.
Dilution integrity
First, a dilution quality control sample (758.78 ng/mL), which was
three times of ULOQ, was prepared in plasma. Then six samples
each of 1/5th (151.75 ng/mL) and 1/10th (75.87 ng/mL) dilution
from the above prepared sample were processed in plasma and
analyzed with freshly processed calibration standards as per the
extraction method. For ATO, % CV were found 1.06 and 1.41 for
1/5th and 1/10th diluted samples, respectively, and % nominal
were found 90.97 and 89.63 for 1/5th and 1/10th diluted samples
respectively. For p-HATO, % CV were found 1.51 and 1.76 for
1/5th and 1/10th diluted samples, respectively, and % nominal
were found 91.84 and 90.00 for 1/5th and 1/10th diluted samples,
respectively. For o-HATO, % CV were found 0.93 and 1.20 for
1/5th and 1/10th diluted samples, respectively, and % nominal
were found 92.47 and 90.79 for 1/5th and 1/10th diluted samples,
respectively.
Stability studies
The stability of ATO, p-HATO, o-HATO, and IS were investigated
in the stock and working solutions, in plasma during storage,
during processing, after four freeze-thaw cycles, at dry extract
stage and in the final extract. Stability samples were compared
with freshly processed calibration standards and QC samples.
Analyte, metabolites, and IS were considered stable when the
percent change of concentration was ±10 with respect to initial
Drug Test. Analysis (2011) Copyright c 2011 John Wiley & Sons, Ltd. www.drugtestinganalysis.com
8. Drug Testing
and Analysis C. Ghosh et al.
Figure 4. (Continued).
Figure 5. Concentration versus time profile of ATO in human plasma from
12 subjects receiving a single oral dose of 80 mg atorvastatin tablet as test
and reference.
concentration. Summary of stability data is presented in Table 3A
for ATO, Table 3B for p-HATO, and Table 3C for o-HATO.
Calibration curve
The plasma calibration curve was constructed using eight
calibration standards (viz. 0.05 to 252.92 ng/mL for ATO, p-
HATO and o-HATO respectively). The calibration curve had a
reliable reproducibility over the standard concentrations across
the calibration range. The calibration curve was prepared by
determiningthebestfitofpeak-arearatios(peakareaanalyte/peak
area IS) vs concentration, and fitted to the y = mx + c using
weighing factor (1/X2). The average regression (n = 3) was found
to be >0.9980. The percentage accuracy observed for the mean of
back-calculatedconcentrationsforthreecalibrationcurvesforATO
was within 94.33–106.84, while the precision (% CV) values ranged
from1.68–6.57; for p-HATO was within 93.20–110.00, while the
precision (% CV) values ranged from 0.01–10.00 and for o-HATO
www.drugtestinganalysis.com Copyright c 2011 John Wiley & Sons, Ltd. Drug Test. Analysis (2011)
9. Determination of atorvastatin and its two positional isomers
Drug Testing
and Analysis
Table 2. Inter- and intra-day accuracy and precision
ATO p-HATO o-HATO
Days Conc. (ng/mL)
Mean Accuracy
(n = 6)
Mean Precision
[% CV] (n = 6)
Mean Accuracy
(n = 6)
Mean Precision
[% CV] (n = 6)
Mean Accuracy
(n = 6)
Mean Precision
[% CV] (n = 6)
Day 1 0.15 109.33 8.54 105.33 6.33 106.00 5.03
26.55 95.27 2.35 92.97 2.70 92.11 2.10
140.35 94.00 2.90 97.68 3.29 96.30 3.11
Day 2 0.15 109.33 3.05 110.00 4.24 104.00 8.97
26.55 94.60 1.05 93.32 1.48 95.29 2.22
140.35 92.32 4.36 96.20 3.40 94.50 4.99
Day 3 0.15 104.67 8.28 108.00 9.88 104.67 8.28
26.55 93.56 2.88 93.11 3.30 93.56 2.88
140.35 93.45 4.04 95.76 2.68 93.45 4.04
Inter Day 0.15 101.74 8.20 94.18 8.73 103.82 7.53
26.55 104.50 5.92 102.37 3.93 107.44 6.57
140.35 99.05 6.15 98.97 6.44 107.26 7.76
Table 3A Summary of stability data of atorvastatin
Stability
QC
level
Mean Precision
(%CV)
Mean
Accuracy
Percent
Change
Stability
Duration
Bench top LQC 3.44 100.28 1.12 6 : 30 Hrs
HQC 1.79 96.86 −0.44
Freeze thaw LQC 0.94 104.17 1.01 4 Cycles
HQC 1.82 96.62 −0.68
Dry extract LQC 2.08 103.24 −1.71 35 : 30 Hrs
HQC 3.99 91.92 −0.75
Auto sampler LQC 3.44 96.06 −2.24 26 Hrs
HQC 7.43 101.23 8.08
Table 3B Summary of stability data of para hydroxy atorvastatin
Stability
QC
level
Mean Precision
(%CV)
Mean
Accuracy
Percent
Change
Stability
Duration
Bench top LQC 2.97 108.93 1.81 6 : 30 Hrs
HQC 2.27 98.76 0.73
Freeze thaw LQC 4.66 105.88 −1.04 4 Cycles
HQC 1.94 97.90 −0.14
Dry extract LQC 3.14 102.76 0.58 35 : 30 Hrs
HQC 4.62 94.04 0.63
Auto sampler LQC 2.94 91.95 −4.40 26 Hrs
HQC 14.84 91.75 1.20
was within 95.51–111.31, while the precision (% CV) values ranged
from 0.23 to 5.69.
Application
The above-described fully validated method was applied to
determine the concentration time profile following single dose
administration of atorvastatin in healthy human volunteers. After
LC-MS/MS analysis the plasma concentration of ATO, p-HATO,
Table 3C Summary of stability data of ortho hydroxy atorvastatin
Stability
QC
level
Mean Precision
(%CV)
Mean
Accuracy
Percent
Change
Stability
Duration
Bench top LQC 3.55 106.12 −0.76 6 : 30 Hrs
HQC 2.79 99.36 0.14
Freeze thaw LQC 1.98 108.16 1.25 4 Cycles
HQC 3.35 98.59 −0.64
Dry extract LQC 5.19 100.29 −4.41 35 : 30 Hrs
HQC 3.12 91.42 −1.05
Auto sampler LQC 5.15 9.42 5.56 26 Hrs
HQC 97.80 97.51 8.71
and o-HATO for all volunteers at times (0.0) and at 0.25, 0.50,
0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 3.00, 4.00, 6.00, 8.00,
12.00, 24.00, 48.00, and 72.00 h for the test (new formulation)
and reference (marketed formulation) products were measured.
The concentration–time profile of ATO, o-HATO, and p-HATO is
presented in Figures 5, 6 and 7. Table 4 represents the detailed
pharmacokinetic parameters of ATO, o-HATO and p-HATO.
Conclusion
A simple, sensitive, selective, precise, and accurate LC-MS/MS
method for the determination of ATO, p-HATO and o-HATO in
human plasma was developed and validated. Unlike the already
published methods, the present method features high sensitivity,
throughput, reproducibility, and precision. Moreover, this method
does not have any matrix effect, such as Ionization abnormalities.
We believe that this method is a useful tool for the determination
of ATO, o-HATO, and p-HATO in human plasma. This method can
be successfully applied for bio-equivalence study of atorvastatin.
Acknowledgement
The authors would like to thank Mr Deepak Rupala, Mr Vijay Jha, Mr
Gaurang Shah,andMsSaumyaBahadurfortheircontributionsand
suggestions for the improvement of the work described herein.
Drug Test. Analysis (2011) Copyright c 2011 John Wiley & Sons, Ltd. www.drugtestinganalysis.com
10. Drug Testing
and Analysis C. Ghosh et al.
Table 4. Pharmacokinetic parameters
ATO o-HATO p-HATO
Test Reference Test Reference Test Reference
Parameter N Mean (±SD)
Cmax (ng/mL) 12 119.92 (±131.91) 64.44 (±58.94) 65.90 (±59.86) 42.89 (±51.99) 14.64 (±17.36) 4.62 (±5.67)
AUC0−t (hr X
ng/mL)
12 383.213 (±324.048) 287.337 (±209.243) 376.915 (±202.205) 288.473 (±182.452) 92.238 (±72.500) 60.910 (±51.683)
AUC0−α (hr X
ng/mL)
12 399.041 (±321.585) 330.549 (±229.001) 389.684 (±201.974) 300.867 (±180.339) 127.915 (±71.405) 115.314 (±48.504)
Tmax (hr) 12 1.458 (±0.916) 1.250 (±1.055) 2.437 (±1.940) 1.854 (±0.956) 6.250 (±4.640) 12.979 (±12.529)
Kel (1/hr) 12 0.127 (±0.044) 0.113 (±0.056) 0.115 (±0.040) 0.100 (±0.037) 0.054 (±0.030) 0.049 (±0.033)
T1/2 (hr) 12 5.998 (±1.833) 8.599 (±6.993) 6.834 (±2.748) 7.976 (±3.4800) 16.608 (±8.605) 24.641 (±22.027)
Figure 6. Concentration versus time profile of o-HATO in human plasma from 12 subjects receiving a single oral dose of 80 mg atorvastatin tablet as test
and reference.
Figure 7. Concentration versus time profile of p-HATO in human plasma from 12 subjects receiving a single oral dose of 80 mg atorvastatin tablet as test
and reference.
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