Determination of Residual Dimethyl Sulfate in Methoxsalen Drug Substance by Pre-Column Derivatization with Static Headspace Gas Chromatography

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 10, Issue 8, 2018

Original Article DETERMINATION OF RESIDUAL DIMETHYL SULFATE IN METHOXSALEN DRUG SUBSTANCE BY PRE-COLUMN DERIVATIZATION WITH STATIC HEADSPACE GAS CHROMATOGRAPHY

AJIT ANERAO*, BHUSHAN PATIL, NITIN PRADHAN R and D centre (API), Wanbury Ltd., EL-16, TTC Industrial Estate, Mahape, Navi Mumbai 400710, India Email: [email protected] Received: 01 Jun 2018 Revised and Accepted: 12 Jul 2018 ABSTRACT Objective: Dimethyl sulfate has been highlighted as potential genotoxic and carcinogenic impurity. A sensitive Headspace gas chromatography (HS- GC) method with pre-column derivatization was developed and validated for the determination of dimethyl sulfate impurity in methoxsalen active pharmaceutical ingredient. Methods: HS-GC method on the column Agilent DB-5, 30m X 0.53 mm, film thickness 1.5 µm, with flame ionization detector (FID) was used. Derivatization reagent concentration, time of reaction and pH of the solution were optimized during method development. This analytical method was evaluated by performing method validation as per ICH guideline. Results: The proposed method was specific, linear, accurate, rugged and precise. The calibration curves showed good linearity over the on (LOD) and limit of quantification (L – concentration range of 0.5 μg/ml to 3.0 μg/ml and the correlation coefficient was 0.999. Method had very low limit of detecti Conclusion: TheOQ) developed 2.0 μg/g method and 5.0 wasμg/g demonstrated respectively. Accuracyto be accurate, was observed robust andwithin sensitive 98.1% for104.5%. the determination of dimethyl sulfate impurity in methoxsalen drug substance.

Keywords: , Dimethyl sulfate, GC-HS, Derivatization, Validation

Methoxsalen http://creativecommons.org/licenses/by/4.0/)

© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license ( DOI: http://dx.doi.org/10.22159/ijpps.2018v10i8.27670 INTRODUCTION methods are found reported of estimation of dimethyl sulfate. Dimethyl sulfate is checked at workplace atmosphere by GC with electrolytic conductivity detector (sulphur mode) at trace level using Methoxsalen is a naturally occurring photoactive substance found in group of compounds known as psoralens or furocoumarins [1]. The workplace atmosphere by thin layer chromatography (TLC) method chemicalthe seeds name of the o Ammi majus (Umbelliferae)-methoxy plant.- -furo It belongs [3,2-g] to[1] a- derivatizingstainless steel with Chromosorb 4- WHP column [13]. DMS is estimated at -one. It has an empirical formula of C12H O4 and a which is very high because TLC technique has limitations in f methoxsalen is 9 1 7H g mole 8 sensitivity point of nitrophenol.view [14]. Quantification The LOQ of DMS of dimethylis reported sulfate 40 ppm by benzopyran7 − anmolecular exogenous weight of 216.19 [2]. Photochemotherapy report suggests that solution stability and diluent study need to be irradiation(PUVA) is treatment [3-5]. involving the use of psoralen, like methoxsalen, opGCMS is reported by derivatizing with pentafluorobenzenethiol. This photosensitizer followed by ultraviolet A (UVA) trace level [15]. Few more methods are reported by extracting the The manufacturing process of methoxsalen is seven step syntheses. timized and mass detector is necessary- to-20]. get DMS sensitivity at by ion chromatography technique with conductivity detector is also used as methylating agent. It is used mainly for converting active- reportedDMS in a solventby separating and tested ionic by GCcompoundsMS [16 usingDMS quantificationAllsep anion In synthetic stage five a key raw material dimethyl sulfate (DMS) is hydrogen compounds such as phenols, amines and thiols to the exchange column [21]. Literature suggests that to achieve such a low corresponding methyl derivatives. Dimethyl sulfate is probably level of limit of quantification hyphenated techniques with . It is a sophisticated instruments like GC- tor or potent genotoxic chemical which can directly alkylate DNA both in LC with conductivity detector is required. It might hardly possible vitrocarcinogenic and in vivoto humans - so IARC classified DMS in Group 2A for quality control laboratory of pharmaceuticalMS or sulphur industry specific detecto perform genotoxicity, the presence of residual dimethyl sulfate in the testing routinely with GC- methoxsalen drug[6 substance8]. Because must of be the controlled known carcinogenicity as per European and maintenance of a mass detector is very high. So in present research, MS because of the cost and drug substance with flame ionization detector. Due to low sensitivity Medicines Agency (EMA), International Conference on ofanalytical FID, premethod-column is developed derivatization to quantify was thetried DMS to in methoxsalenachieve the “thresholdHarmonization of toxicological [9] and Foodconcern” and (TTC) Drug concept Administration for the limit (FDA) of quantification of analyte at trace level. The proposed analytical genoguidelines [10, 11]. EMA and FDA guidelines proposed the use of the method was validated as per International conference on harmonization guidelines ICH Q2-R1 [22]. day totoxic/carcinogenic the expected dose impurities. of drug The substance concentration in a gram limit, per in ppm,day. of genotoxic impurity in drug substance, is the ratio of TTC in μg per MATERIALS AND METHODS methoxsalen per day [1, 12], dimethyl sulfate must be limited to less Considering the recommended daily maximum dose of 0.070 g Method of analysis sensitive, accurate and robust analytical method. Instrumentation than 21.4 μg/g in drug substance. So it is necessary to develop -500 GC with headspace auto sampler and flame ). Dimethyl sulphate is not controlled in the ionization detector (FID) using Total Chrome Navigator software. Methoxsalen is official drug substance in United State ReferPerkin chromatographic Clarus parameters as per given in table 1. Pharmacopoeia (USP USP monograph [2]. During literature survey several analytical

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Table 1: Chromatography parameter Column DB-5, 30m X 0.53 mm, Film Thickness: 1.5 µm Oven Temperature 40 °C 40 - - hold for 5 minute, DetectorOven Program Flame°C hold Ionization for 8.0 Detectormin, ramp (FID)1 at 5 °C/min to 100 °C hold for 0 minute, ramp 2 at 20 °C/min to 250 °C Detector temperature 220 °C Carrier Gas Nitrogen280 °C CarrierInjector Gas temperature pressure 5.0 psi Run Time 32.50 min Headspace Conditions Oven Temperature Sample Line Temperature 110 °C Transfer line Temp 12090 °C °C

3.0 min Headspace Pressure 0.2016 psi min WithdrawPressurize timetime 0.5 min ThermostatInject time time 30.0 min GC Cycle Time

46.0 min Chemical and reagents followed by two test preparations. Run the chromatograph for 32.5 min and record the chromatogram. The retention time of dimethyl GC columns used of Agilent DB-5 of a dimension 30 meters, internal sulfate derivative i.e. anisole is 15.0 min. diameter 0.53 mm with film thickness 1.5 µm. Agilent DB-1 of a dimension 30 meters, internal diameter 0.53 mm with film thickness Acceptance criteria for system suitability 3 µm. Agilent DB- internal diameter 0.53 mm with film thickness 3 µm. Anisole standard was procured 624 of a dimension 30 meters, should not be more than 15.0. % RSD of area of dimethyl sulfate peak in six replicate injections methyl sulfoxide from Rankem, phenol from CALCULATION from AVRA synthesis of purity 99.5%, dimethyl sulfate from Finar Ltd.water of was purity used 99.0%, in the di experimentation. Calculated the content of dimethyl sulfate in ppm by formula given Rankem, sodium hydroxide from Merck and purified HPLC grade below Solution preparation – Diluent preparation: phenol and 5.0 g of sodium hydroxide into 500 ml volumetric flask (peak area of DMS6 in sample peak area DMS in blank) × Conc. of std. and diluted up to the markAccurately with premixed weighed dimethyl and transferred sulfoxide: 10.7 Water g of in mg/ml x P x 10 – minute again sonicated for two minutes. This solution is used as (Avg. peak area of DMS in std. peak area of DMS in blank) × Conc. of diluent(1:1 v/v) for mixture. preparation It was of blank,sonicated standard for two solution minutes; and shaketest solution. for one sampleRESULTS in mg/ml x 100 Analytical method development Standard stock solution A: Accurately weighed and transferred 0.10 g of dimethyl sulfate standard into 100 ml volumetric flask containing 40 ml of diluent and diluted up to the mark with diluent, has no chromophores. For analysis of such a ultr- shake it for two minutes again sonicated for five minutes. volatileDimethyl compoundsulfate is a volatilegas chromatography compound of boiling (GC) pointis a188 ⁰Csuitable and technology. To quantify the analyte at parts per millionviolet (UV)(ppm) inactive levels, Note: 1) Diluent should be ready before weighing of dimethyl sulfate standard. the concentration of test solution is required more. If the large 2) After transfer of dimethyl sulfate standard to 100 ml volumetric term usage so head space technique is preferable over liquid flask immediately dilute up to the mark with diluent. quantity of test solutionity of flame is injected ionization on GC detector column itis may not spoilsufficient in long to detect dimethyl sulphate at such a low concentration. So to enhance Accurately transferred 1.0 ml of Standard stock solution B: theinjection. response Sensitiv of dimethyl sulphate it was decided to perform pre- standard stock solution A in to 100 ml volumetric flask, added column derivatization technique. Considering all above properties of diluent and diluted up to the mark with diluent. Shake and mix well. the compound and purpose of the requirement of method Standard stock solution C: Accurately transferred 5.0 ml of development, GC headspace pre-column derivatization method is standard stock solution B in to 25 ml volumetric flask, added diluent developed with FID after optimizing chromatographic parameters. and diluted up to the mark with diluent. Shake and mix well. reported in literature [23]. The derivatized complex of dimethyl Blank preparation: In to a 20 ml GCHS vial added 2.0 ml of diluent sulphateMethylation and of phenol phenol is withanisole DMS which in presence has considerable of sodium and hydroxide sufficient is and crimped the head space vial with septa, star spring and alumina response with FID. cap. Standard solution: In to a 20 ml GCHS vial added 2.0 ml standard solution It has been reported that DMS can be degraded stock solution C and crimped the headspace vial with septa, star with sodium hydroxide solution (1 mol/l), sodium carbonate spring and alumina cap. with water(1 mol/l),(methanol, or ammonium ethanol, hydroxidedimethyl solutionsulfoxide, (1.5 dimethyl mol/l). formamide,Complete destruction acetone) ofor undilutedsolvents DMSpartially or DMS miscible in solvents or immiscible miscible Test preparation: Accurately weighed and transferred 0.2 g of a with water (toluene, p-xylene, benzene, 1-pentanol, ethyl acetate, sample in to a 20 ml GCHS vial and added 2.0 ml of diluent and chloroform, carbon tetrachloride, acetonitrile) [24]. So many crimped the headspace vial with septa, star spring and alumina cap. observed that further after derivatization the area of anisole was Procedure foundattempts inconsistent were made toand stabilize reducing the DMSgradually. in various The solvents derivatization but it is reaction mechanism is explained in fig. 1.

Performed blank followed by standard solution six times, blank

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OH O dimethyl sulfate standard peaks are eluting exactly at same NaOH Na retention time 15.0 min. During method optimization various stationary phases of different polarity were used like DB- - dimethylpolysiloxane) and DB- 624 (6% phenol sodium phenoxide dimethylpolysiloxane).cyanopropyl phenyl and In 94%DB- dimethylpolysiloxane), DB 1 (100% O O - 5 (5% diphenyl and 95% (CH ) SO Na 3 2 4 CH3 than 2.0. DB- 624 DMS was eluting very close to DMSO peak and with DB 1 column DMS peak tailing factor was more symmetrical. 5 column was selected because DMS peak was separated from DMSO with resolution more than 5.0 and peak was sodium phenoxide anisole Analytical method validation

Fig. 1: Derivatization reaction mechanism and formation of Selectivity anisole Selectivity is the ability of the method to determine accurately and specifically the analyte of interest in the presence of other This is two step reactions and it gets complete at room temperature. components in a sample matrix that may be expected to be present Once the sodium phenoxide is formed it is found stable. So first in the sample matrix under the stated conditions. Selectivity of the sodium phenoxide is prepared by addition of phenol and sodium method was evidenced by comparing blank, dimethyl sulfate, hydroxide at room temperature in aqueous dimethyl sulfoxide solution. This solution is used as a reagent blank, for preparation of is no interfering peak at the retention time of dimethyl sulfate is obsermethoxsalenved. Dimethyl and all sulfate specified is wellimpurities resolved separate from all injections. other specified There anisole derivative was found stable in solution form and further the solvent peaks which are used in the manufacturing process of methodDMS standard is found and satisfying test solution. all validation After optimization parameters of these like stepslinearity, the methoxsalen. Selectivity of methanol, acetone, methylene chloride, solution stability and accuracy. Anisole reference standard was n-hexane, ethyl acetate, toluene and benzene had been checked and found separated from dimethyl sulfate peak. Refer figure-2 and 3 of analysis. The retention time of anisole standard and derivatized dimethyl sulfate standard solution and test solution chromatograms. procured from Avra Synthesis and injected in proposed method of

Fig. 2: Dimethyl sulfate standard solution chromatogram

Fig. 3: Test solution chromatogram of methoxsalen

Solution stability - DMS standard solution was prepared fresh before injection and function of storage conditions and chemical properties of the drug immediately injected and same solution was injected after twentyt was andDrug its stability impurities. in the The active solution pharmaceutical stability till ingredients twenty-six (API)hours is of a six hours. The peak area of DMS of freshly prepared standard-six solution. hours.solution was observed 280527 and after twenty six hours i 272834. No significant change in area was observed after twenty derivatized DMS had been checked by injecting standard

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LOD and LOQ LOD and LOQ concentration are reported in parts per million (ppm) with respect to methoxsalen test concentration. LOD is 2.0 ppm and The limit of detection (LOD) is the lowest concentration of the analyte in a sample that can be detected but not necessary quantified. The obtained LOD values of LOQ is 5.0 ppm. Precision of LOQ is checked by injecting six replicate is discussed. specified impurities and API consistencyinjections. Relativeand reproducibility standard of deviation the method (RSD) at trace of peaklevel. area of dimethyl sulfate at LOQ level was observed 5.4% which proves Linearity TheLOD =limit 3.3 ×of σ/S quantitation (LOQ) is the lowest concentration or amount of the analyte that can be determined quantitatively within imental conditions, the peak area vs. concentration an acceptable level of repeatability precision and trueness. plot for the proposed method was found to be linear over the range Under the exper LOQ = The regression coefficient (r2) of LOQ level i.e. 25%, 50%, 100%, and 150% of the specified limit 10.0 × σ/S calibration curve withfit of thea regression data to the coefficient regression is line.0.999. Linearity plot is given in fig. 4. Where σ = the standard deviation of the response and S= slope of the is more than 0.99 is generally considered as evidence of acceptable

Fig. 4: Linearity plot of dimethyl sulfate derivative

Accuracy Ruggedness study Accuracy can be defined as the closeness of agreement between The intra-laboratory tested behavior of an analytical process when small a test result and the accepted reference value. Accuracy of the changes i method was determined by recovery study. Analytical method may be considered validated in terms of accuracy if the mean n the environment and/or operating condition are made. The different days. Validation batches were prepared by each analyst value is ruggedness of the method was evaluated by estimating % RSD of derivatized DMS standard solution tested by two different analysts on

within±20% of the actual value. During recovery study separately. % RSD of area of DMS peak in standard solutions of both themethoxsalen limit of dimethyl API batch sulfate was analyzed that is 21.4 and ppm.then dimethylThe recovery sulfate was is analysts should not be more than 10%. Six replicates of diluted standard foundspiked 104.5 in the API at LOQ level, 100% and 150% with respect to solution were injected by each analyst. Relative standard deviation of the method is capable to quantify the analyte accurately and area of dimethyl sulfate was found 1.02% and 5.06% on two different results will be%, reliable 93.8% andeven 98.1% at trace respectively level. which proves that days. Content of dimethyl sulfate in one API batch was tested on two different days and the results were found 1.6 ppm and 1.3 ppm respectively. Method validation summary is given in table 2. Table 2: Analytical method validation summary S. No. Parameter Acceptance criteria Results 1 Selectivity peak should be well separated from all known and unknown impurities of methoxsalen. 2 Solution stability ReportDMS derivative Result SolutionsMethod is areselective stable up to 24 h 3 Limit of detection Report Result 2.0 ppm wrt test concentration 4 Limit of quantification Report Result 5.0 ppm wrt test concentration 5 LOQ precision RSD 5.4 Linearity R2 Accuracy Recovery%RSD for shouldsix replicates be between of LOQ level standard solutions is NMT: 10.0% % 6 Correlation: NLT 0.99 0.999 7 RSD98.1% 1.02 to 104.5% Intermediate precision 80% to 120% RSD 3.5 8 Method precision %RSD for results of six standard replicate is NMT: 10% % 9 %RSD for results of twelve standard replicate (Method precision and % Intermediate precision) is NMT: 10% ive standard deviation, LOQ is limit of 2 is correlation coefficient. Abbreviations: DMS is a dimethyl sulphate, h is hour, ppm is parts per million, wrt is with respect to, %RSD is percent relat quantification, NMT is not more than, NLT is not less than and R

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DISCUSSION REFERENCES Dimethyl sulfate is used in the manufacturing process of 1. Rx Available from: methoxsalen. Since the dimethyl sulfate is known mutagenic and -drug.htm. [Last accessed on 01 carcinogenic impurity it should not be detected in the medicinal List. Uvadex. Methoxsalen. 2016; product. As per ICH guideline the allowable intake of dimethyl 2. http://www.rxlist.com/uvadex sulfate is calculated considering maximum daily dose of May 2018] 201 - methoxsalen. So the limit of dimethyl sulfate was found 21.4 ppm. 3. ShenoiUnited StateS, Pharmacopoeia. Methoxsalen official monograph. andUSP vitiligo.40; 6.Ind p.ian 5087 J Derm8. Vene Leprol 201 0: -504. 4. Prabhu S. 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So fresh or immediate Risk Chem Hum 24:1–33 . 1977;68:328 testing of the derivatized product was not required. To make the 6. IARC. GeneticSome pharmaceutical and related effects. drugs. An IARC updating Mon of selected IARC stable complex, first phenol was reacted with sodium which gives 1980; 7 r Eval Carcinog sodium phenoxide and this is used as diluent for preparation blank, 7 Risks Hum Suppl 1– . standard, test and all other solutions for validation study. Sodium . IARC.monographs Overall from evaluations Volumes of1 tocarcinogenicity. 42. IARC Monog An updating of phenoxide is quite stable at room temperature that is later on 1987;6b: 729 8 1–440. -six . IARC Monographs volumes 1 to 42. IARC Monogr Eval Carcinog hours.reacting The with stability DMS of to the form derivatized stable anisole. complex Solution is further stability confirmed was Risks Hum Suppl. 1987;7a: duringchecked method and found validation that the recovery DMS derivative study. It wasis stable perfo tillrmed twenty at three 9 ICH. M7(R1) Addendum to ICH M7. Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals-Specific to AcceLimit Potential Carcinogenic Risk; Application of the Principles derivatizeddifferent levels complex from is LOQvery tostable 150%, and and quantifiable recovery at was such observed a trace 10. ICH.of the Impurities ICH M7 in Guidance new drug to sub Calculationstances of Compound level.minimum The method 98.1% andwas maximumso sensitive 104.5% that it could which detect proves th e that analyte the 11. ptable Intakes. ICH Harmon Guid; 2017. even at 2 ppm and can be quantified at 5 ppm which sufficient and Guidance for Industry ANDAs Q3A (R2); 2006. FDA. U. S. Dept of Health and Human Services Food and Drug Adm. Linearity study indicated that area was directly proportional to 12. : Impurities 201 in Drug Substances; concentrationvery low level comparedof the derivatized to the limit analyte of the DMSwith genotoxic R2 impurity. 2009. [Last accessed Drugs. com. Methoxsalen dosage; 6. Available from: the analyte. The method was further challenged with inter0.999 and in intra the- . https://www.drugs.com/dosage/methoxsalen.html. laboratoryconcentration changes range ofin LOQ operation to 150% parameters level with respectwhich toare the possible limit of 13. . on 01 May 2018] during routine day to day testing as well as during technology 4. Available from: NIOSH. Manual of Analytical Methods (NMAM).- Fourth Editionpdf. confirmed by performing the testing on different days and two DIMETHYL SULFATE. METHOD 2524; 199 differenttransfer analysts to another with laboratory.separate instruments Precision and of found the resultsthat relative was 14. FEAIIG.https://www.cdc. Federationgov/niosh/docs/2003 of the Employment154/pdfs/2524. Accidents Insurance is the indication of [Last accessed on 01 May 2018] reproducibility of the method at given laboratory conditions. dimethyl sulfate standardFurther, the deviation method waswas lesssuccessfully than 10% applied that to three methoxsalen Institutions of Germany. Centre for Accident-105. PreventionAvailable from: and validation batches with dimethyl sulfate concentration levels were Occu Med. Method for the determination of found far below the LOQ. (DMS). Ana Met Air Monit Met 1997;5:75 15. http://onlinelibrary.wiley.com/doi/10.1002/3527600418.amTrace level determination of residues of dimethyl sulfate CONCLUSION 7778e0005/pdf.and diethyl sulfate [Last in pharmaceutical accessed on 01 products May 2018] by headspace gas In conclusion, quantification method was developed to check chromatograpPrabhu P. hy and mass spectrometry following derivatization genotoxic process impurity dimethyl sulfate in methoxsalen drug substance. The proposed method was validated as per ICH guideline 2015. Available from: - and checked accuracy and reliability of the result. The static head with pentafluorobenzenethiol.-Trace Experimental-level-determination Notes Perkin-residues Elmer; space GC-FID analytical method with pre-column derivatization of dimethyl sulfate.pdf. [ http://www.perkinelmer.com/ CMSRe satisfies all validation parameters like system suitability, precision, . sources/Images/20130104_8Zheng J, Wayne A, Zhang S, Wittenberger S. Determination of specificity, accuracy, linearity of detector response, ruggedness and Last accessed on 01 May 2018] robustness. It indicates that the method is stable and suitable for the 16 intermediate using liquid–liquid extraction and GC– J quantification of dimethyl sulfate in methoxsalen. Hence, the low ppm levels of dimethyll 200 5 0:1054sulfate -in an aqueous soluble API validated method can be used for routine analysis of quantification . Nalavade A, Sandhyakumari B, Ramakrishna K, SrinivasaraoMS. V. of dimethyl sulfate in methoxsalen in quality control laboratories in PharmaDevelopment Biomed and Ana evaluation9; of 9.class-1 solvent 1, 2- the pharmaceutical industry. 17 dichloroethane and dimethyl sulphate as a genotoxic impurity in sildenafil citrate drug substance by using GC- technique. ACKNOWLEDGEMENT 201 -5. Thanks to the management of Wanbury Limited for constant . Schäfer C, mination of trace levelsMS of dimethyl support. sulfateInt J Pharm in Pharmathe presence Sci 4;of6;552 monomethyl sulfate by gas 18 chromatographyZöllner-mass P. Deter spectrometry. J Chrom A 201 10. AUTHORS CONTRIBUTIONS . Rahman Gullick D, Hoerner J, Bartlett Determination of 3;4: All authors contribute equally to this manuscript genotoxic impurities monomethyl sulfate and dimethyl sulfate in 19 active pharmaceuticalM, ingredients. Anal M. 201 - CONFLICT OF INTERESTS 20. Nanduri V, Reddy K, Adapa V, Ramakrishna K. Validated chromatographic methods for the determinationMethods of process7;7:1112 related8. toxic impurities in pantoprazole sodium. Chromatographia they have no conflict of interest. Author Ajit Anerao, Bhushan Patil and Nitin Pradhan declare that 200 : –4. Ethical approval: This article does not contain any studies with 21. human participants or animals performed by any of the authors. 8;68 481 et al. 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chromatography method for the simultaneous determination of 23. Hiers G, Hager F. Anisole. Org Syn 5 12. dimethyl sulfate and diethyl sulfate contents in metoprolol Doi: 2 tartrate drug substance. Chem Sin 201 - 24. Lunn G, Sansone E. Validation of 194techniques1;1; 8;9; 192for 9;the 22. ICH. Validation of analytical procedures: Text and methodology destruction10.15227/orgsyn.009.001 of dimethyl sulfate. Am Indu Hyg Assoc J Q2 (R1) 2005. 1;2:164 72. 4 -4.

; 1985; 6:111