Chiaramente, ogni formato ha i propri vantaggi e svantaggi comprare doxycycline senza ricetta per effettuare un acquisto, non è necessario fornire la prescrizione medica.

10337_2009_954_article 1.5

LC Determination of Isosorbide-5-Mononitrate in Human Plasma Himanshu S. Karmalkar&, Mohan M. Metku, Milind S. Bagul, Asmita C. Nimkar, Rajen D. Shah Raptim Research Limited, A-226, TTC Industrial Area, Mahape, Navi Mumbai, Maharashtra 400701, India; E-Mail: hskarmalkar@gmail.com Received: 3 June 2008 / Revised: 23 November 2008 / Accepted: 15 December 2008 LC-MS–MS [–] and GC–MS []However, no LC–UV method has been developed for the determination ofISMN from human plasma.
A simple, sensitive, selective and cost effective LC–UV method was developed for determi- GC-ECD is the most widely used nation of isosorbide mononitrate in human plasma using guaifenesin as an internal standard.
instrumental method due to its high Isosorbide mononitrate in plasma was extracted by a single step liquid extraction using tert- sensitivity and selectivity. However, it butyl methyl ether and chromatographed on a C18 column using water and acetonitrile was shown by Pastera et al. that (80:20 v/v) as mobile phase. The method was validated and exhibited a linear range from ISMN had a substantial decomposition 51.6 to 2064.4 ng mL-1. The inter- and intra-assay accuracy ranged from 97.2–102.7 to when the injector temperature was higher 94.2–105.5%, respectively, with precision less than 10% in both the cases. The LLQ was than 150 °C. If the injector temperature 51.6 ng mL-1. The validated method was applied to the quantitation of isosorbide mononi- was low, it promoted the condensation of trate from plasma samples in a pharmacokinetic study.
non-volatile compounds causing con-tamination of injector system and a rapiddecrease in the sensitivity. Morrison et al.
[] used packed GC columns withoutmononitrate derivatisation.
SPE with ENVI 18 cartridges ] or Column liquid chromatography Extrelut columns ] replaced the labo- rious liquid–liquid extraction in three Isosorbide-5-mononitrate in human plasmaGuaifenesin methods. Gremeau et al. ] obtained aLLQ of 20 ng mL-1. Pommier et al. []reported a method requiring a silylationpretreatment of glassware to preventadsorption of ISMN to the glass. Jain longer elimination half-life, no first-pass et al. [quantified ISMN via stable metabolism and no metabolites, leading acetate adduct formation using LC-ESI- to more predictable and reproducible MS–MS to give a LLQ of 10 ng mL-1.
clinical effect The salient features of their method were [3.3.0] octan-4-ol is an active metabolite The analytical techniques developed low sample volume and SPE with direct of isosorbide dinitrate. ISMN is a long in recent years for the quantitation of injection of eluent and short run time.
acting organic nitrate vasodilator used in ISMN from human plasma include LC The method described here was the treatment of angina pectoris. It has with a Thermal Energy Analysis detector developed with the aim of obtaining a [], capillary GC with an electron- compound isosorbide dinitrate, such as effective LC–UV analysis with a short Full Short CommunicationDOI: 10.1365/s10337-009-0954-x Ó 2009 Vieweg+Teubner GWV Fachverlage GmbH run time for the determination of ISMN EZChrome Elite software (version-3.2.1) was 22%. Interference from plasma at in human plasma. tert-Butyl methyl was used for data handling. Analysis was the retention time of ISMN was ob- ether as extraction solvent evaporates performed on a 5 lm Merck Purosphere served in samples extracted with diethyl very quickly, allowing 100 samples to be ether. tert-Butyl methyl ether was the processed and analysed in a day. The solvent which showed no interference at extraction efficiencies of ISMN using with water:acetonitrile (80:20 v/v) at the retention time of ISMN and GF and tert-butyl methyl ether in polypropylene 1 mL min-1. The column was main- yielded an extraction efficiency of better and glass tubes were found to be similar tained at 20 °C. Detection was carried than 50%. An appropriate internal and consistent, indicating that there was out at 220 nm.
standard can control extraction and LC no loss by evaporation of nitrates or injection variability. Although Guaifen- sorption to glassware. ISMN is a very esin is not chemically and structurally Preparation of Stock related to the analyte, it was chosen as hence our method is not as sensitive as and Working Solutions an internal standard for its stability in other reported methods. Although the prepared solutions and a good response LLQ value of 51.6 ng mL-1 established Primary stock solutions of ISMN and at the detection wavelength. Its selection in our method was higher it is regarded GF were separately prepared by dissolv- was also based on its good chromato- as acceptable. The method was success- ing an appropriate amount in methanol graphic and extraction behavior.
to yield a concentration of 1 mg mL-1 of pharmacokinetics of ISMN after oral each. The standard working solutions administration of 60 mg sustained re- were obtained by further dilution of the Sample Preparation lease (SR) tablets to healthy volunteers.
stock solutions with mobile phase. TheGF working solution had a concentra- Calibration standard samples and qual- tion of 25 lg mL-1. All solutions were ity control samples were extracted by stored at 2–8 °C and equilibrated to using liquid–liquid extraction. To each room temperature before use.
polypropylene vial containing 0.5 mL Chemicals and Reagents sample, 50 lL of IS was added at aconcentration of 25 lg mL-1 and also3.0 mL of tert-butyl methyl ether, which All chemicals and reagents used in the Preparation of Calibration were then vigorously vortex-mixed for study were LC grade. Acetonitrile and Standards and Quality Control 3 min followed by centrifuging at 4,500g tert-butyl methyl ether were purchased at 4 °C for 5 min. The organic phase was from Merck (Mumbai, India). Water transferred to a new polypropylene vial used in the mobile phase was deionised Calibration standards were prepared at and evaporated to dryness in a water and purified by a Milli-Q water purifi- 8 different concentrations of 51.6, 103.3, bath at 40 °C under a stream of nitrogen cation system from Millipore (Bedford, 206.6, 413.1, 826.2, 1238.6, 1651.5 and gas. The dried residue was reconstituted MA, USA). A working standard of 2064.4 ng mL-1 of ISMN in plasma.
with 100 lL of mobile phase and vortex- ISMN was obtained from Pan Drugs Quality control (QC) samples were pre- mixed briefly. A 50 lL aliquot was in- (Gujarat, India), and guaifenesin (GF) pared at three different concentrations jected into the LC system.
which was used as an internal standard of low QC (LQC-151.7), medium QC was obtained from Unique Pharmaceu- tical Labs. (Thane, India). Drug free 1816.7) ng mL-1. All of these samples Method Validation human CPDA (citrate phosphate dex- were prepared by spiking appropriate trose adenine) plasma bags (six different amounts of standard stock solutions into The method validation was carried out lots) were obtained from JVP Blood the drug free plasma samples. They were according to the US Food and Drug Bank (Vashi, Navi Mumbai, India).
stored at -20 °C.
validation guidance [].
The specificity of the method was Method Development tested by screening six different batches of drug free human plasma. Each blank Dichloromethane, diethyl ether, n-hex- sample was tested for interferences at the The LC instrument was an Agilent ane, tert-butyl methyl ether and their retention time of ISMN and GF using 1200 series LC (Waldbronn, Germany) mixtures with each other were tried for the proposed extraction procedure. Fre- the extraction of ISMN and GF. With quently used over-the-counter medicines such as acetaminophen, ibuprofen, dic- Peltier cooler (G1330B), thermostatted ciency of the analyte was 12%. With a lofenac and cetirizine were also injected column compartment (G1316A) and a mixture of dichloromethane and n-hex- to check for interference at the retention variable wavelength detector (G1314B).
ane (1:1 v/v) the recovery of the analyte time of the analyte and GF. For sensi- Full Short Communication tivity determination, six samples at LLQ concentration (51.6 ng mL-1) of ISMN were investigated and the reproducibilityand precision were determined.
Linearity was tested for the concen- tration range of 51.6–2064.4 ng mL-1.
For the determination of linearity, ablank sample, a zero sample (blank+ IS) and eight calibration standards were used. Samples were quantifiedusing the ratio of peak area of analyte to that of the IS. A weighted linear regres- sion (1/concentration) was performed with nominal concentrations of calibra- tion levels. Peak area ratio was plottedagainst standard curves were calculated.
The intra-day precision and accuracy of the assay were measured by analyzing six spiked samples of ISMN at each QC level (151.7, 908.3 and 1816.7 ng mL-1).
The inter-day precision and accuracy was determined over 3 days by analyzing30 QC samples (n = 6 for each concen- tration level) each day.
The validation was carried out using Fig. 1. Representative chromatograms of ISMN (1) and guaifenesin (2) in human plasma.
human plasma which contained CPDA A Blank human plasma; B blank plasma spiked with ISMN and IS at LQC level; C plasma as anticoagulant. Normally EDTA is sample from a subject 4 h after oral administration of 60 mg sustained release (SR) isosorbide-5- used as an anticoagulant and it was mononitrate formulation therefore necessary to assess the antico-agulant effect and understand its impacton the quantitation of ISMN from aplasma matrix. A significant anticoagu- ples were kept in the autosampler at 7 °C respectively. The total analysis time was lant effect would prevent using EDTA as for 24 h and then injected to determine an anticoagulant. Anticoagulant effect the autosampler stability The freeze– No interfering peaks were observed at was performed by comparing LQC and the retention times of either the analyte or HQC samples (n = 3) prepared in EDTA -20 ± 5 °C), was studied after subject- IS in six different batches of drug-free anticoagulant-based human plasma and ing the samples to three freeze–thaw human plasma samples. A representative cycles. For dry extract stability the dried LC chromatogram of extracted blank prepared in CPDA anticoagulant-based extracts were kept at 2–8 °C for 24 h plasma is shown in Fig. The precision after which they were brought to ambi- for the six plasma samples spiked with Evaluation of stability of samples ent temperature, reconstituted and then ISMN at LLQ concentration was 5.9% was based on the comparison of stored analyzed. In order to determine the sta- with accuracy ranging from 98.6 to samples against freshly prepared samples bility of ISMN in solution, the working of the same concentration. The percent- solution was kept at 2–8 °C for 6 days.
The method exhibited a good linear age difference between the back calcu- Thereafter, the mean areas of ISMN response over the range of concentra- lated concentrations obtained in the from six replicate chromatographic runs tion from 51.6 to 2064.4 ng mL-1. The stored samples and freshly prepared were compared to that of mean area of a mean correlation coefficient was 0.998.
samples was evaluated. Four aliquots freshly prepared solution of the same The mean accuracy values of each cali- each of LQC and HQC concentrations bration standard were close to each were used for the stability study. For assessment of long term stability, the samples were kept in deep freezer at Results and Discussion between 94.9 and 105.5% with a precision -20 ± 5 °C for 30 days and thereafter of 6.9–9.2%. The inter-day accuracy was analyzed. Bench top stability was studied Using the chromatographic conditions between 97.2 and 102.7% with a precision on samples kept at ambient temperature described above, the peaks of ISMN and of 5.4–9.9%. The results are presented in (20–30 °C) for 8 h. The processed sam- GF eluted at about 3 and 6 min, Table A chromatogram of ISMN at the Full Short Communication Table 1. The intra- and inter-batch precision and accuracy of the method for the determination found to be stable for 6 days at 2–8 °C of ISMN in human plasma with %RSD of 1.2%.
(mean ± SD, ng mL-1) Pharmacokinetic Study Figure shows the time course of the ISMN plasma concentration of 21 heal- thy human subjects who received 60 mg sustained release ISMN tablets under conducted strictly in accordance withguidelines laid down by the International USFDA []. The pharmacokinetic data are tabulated in Table -1 Lm. 400
The developed and validated LC method proved to be cost effective, simple, rapid, selective and convenient for the deter- mination of ISMN in human plasma.
The method has the advantages of goodselectivity, procedure, high throughput and wide linear range. It has been successfullyapplied to the pharmacokinetics of Fig. 2. Time course of the plasma ISMN concentration in healthy subjects after a single 60 mg ISMN in healthy volunteers after single (SR) oral dose. Each point represents the mean ± SD (n = 21) oral administration of 60 mg sustainedrelease formulation.
Table 2. Pharmacokinetic parameters of ISMN in 21 healthy subjects after a single 60 mgsustained release oral dose 1. O'Neil Maryadele J (ed) (2001) The Merck index, 13th edn, monograph no.
5246, p 935 and monograph no. 4571, p 812. Merck Research Laboratories, Division of Merck, Whitehouse Station 2. Chasseaud LF (1987) Cardiology 74:6.
LQC concentration with an internal 2.7% for LQC and HQC, respectively, 3. Major RM, Taylor T, Chasseaud LF, Darragh A, Lambe RF (1984) Clin standard in shown in Fig.
for 8 h at ambient temperature with Pharmacol Ther 35:653 The accuracy and precision obtained percent difference of 8.0 and -4.5% for 4. Pastera J, Vyslouzil L, Kvetina J (2004) J on LQC samples prepared using EDTA LQC and HQC, respectively, for 24 h in Chromatogr B Analyt Technol BiomedLife plasma ranged from 98.6 to 102.0% with the autosampler with percent difference precision of 2.3 and 99.6% to 101.3% of 12.4 and -1.0% for LQC and HQC, 5. Gremeau I, Sautou V, Pinon V, Rivault with a precision of 1.0% on HQC sam- respectively. The dried extract was stable F, Chopineau J (1995) J Chromatogr B ple indicating that there was no antico- for 24 h at 2–8 °C with percent differ- Analyt Technol Biomed Life Sci 665:399–403. agulant effect and the biostudy samples ence of 2.6 and -1.8% for LQC and 6. Michel G, Fay L, Prost M (1989) J could be collected using EDTA as an HQC, respectively and ISMN in plasma Chromatogr A 493:188–195 was stable for three freeze–thaw cycles 7. Santoni Y, Rolland PH, Cano J-P (1984) J Chromatogr A 306:165–172 ISMN was found to be stable in the with percent difference of 9.2 and plasma matrix for 30 days at -20 °C -13.6% for LQC and HQC, respec- Chromatogr A 308:153–164. (LTS) with percent differences of 4.6 and tively. The stock solution of ISMN was Full Short Communication 9. Maddock J, Lewis PA, Woodward A, Massey PR, Kennedy S (1983) J Chro- Chabard JL, Beyssac E, Aiache JM, matogr A 272:129–136 14. Jain DS, Subbaiah G, Sanyal M, Shriva- Nadesclaire M (1995) J Chromatogr B 10. Marzo A, Treffner E (1985) J Chroma- stav PS, Pal U, Ghataliya S, Kakad A, Analyt Technol Biomed Life Sci 663:153– togr A 345:390–395. doi: Bhatt J, Munjal V, Patel H, Shah S (2006) Rapid Commun Mass Spectrom 20(19): 11. Pennings JM, De Haas JM (1995) J 2921–2931. doi: Method Validation, Center for Drug Chromatogr B Analyt Technol Biomed 15. Silva LC, Oliveira LSOB, Mendes GD, Evaluation and Research (CDER), May Life Sci 675:332–336 Garcia G, Pereira ADS, Nucci GD (2006) 12. Sioufi A, Pommier F (1984) J Chroma- J Chromatogr B Analyt Technol Biomed 19. Guidance for Industry, E6 Good Clinical togr A 305:95–103 Practice: Consolidated Guidance, Center 13. Pommier F, Gauducheau N, Pineau V, Sioufi A, Godbillon J (1996) J Chroma- 16. Sun X, Li X, Cai S, Qin F, Lu X, Li F (CDER), April 1996 togr B Analyt Technol Biomed Life Sci (2007) J Chromatogr B 846:323–328 Full Short Communication

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