__BIOPHARMACY THE INFLUENCE OF RELATIVE HUMIDITY AND TEMPERATURE ON STABILITY OF MOEXIPRIL HYDROCHLORIDE IN SOLID PHASE BEATA STANISZ Poznati, University of Medical Sciences, Department of Pharmaceutical Chemistry, 6 Grunwaldeka Str, 60-780 Poznati, Poland Autrac: Kinetic and shennedynamie parameters ofthe decomposition of roexpi ysl in solid phase inthe absence and presence of humidy were cslelated. The evalua of viability of mex hyirochloride was fllowed by the HPLC metho. The applied thou wax valid (valaton of the following parameter: seletity. linear pein, init of detection LOD). nto guns (LOQ) and eyetailiy) The eff banity he tbl of MCL ns phase at 368K was describe bythe ution: nk = a + b= (0.0675 + 0.016) RH ~ (15.83 0.98. Mention of deradton products IMHCI were cared out by the HPLC-MS metho Keywords: mossiprilhydroclorde (MICH: slit phase; HPLC; HPLC-MS; kinetic and thesmexy nami Moexipril_ hydrochloride (MHCI)((38}-2: [(25)-N-[(18)~1-catboxy-3-phenylpropylalanyl) =1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquino- linecarboxylic acid, 2-ethyl ester) belongs. to 4 class of N-carboxyalkyl dipeptide angiotensin converting enzyme (ACE) inhibitor. Moexipril is a prodrug. which is hydrolyzed after absorption to the active diacid moexipril (moexiprilat). Both ‘moexipril and moexiprilat inhibit ACE activity, although the potency of moexipril is much lower than that of moexiprilat. Moexipril and moexip- rilat inhibit ACE activity and thereby prevent the formation of angiotensin I from angiotensin I a3) The effect of organic solvents on the de- gradation kinetics, onthe product formation and on the mechanism of MHCI transformation at 25 hhas already been investigated and the results are presented in the paper of Gu L, Strickley R.G. eta (4.5). The stability of MHCI was also studied in the presence of selected excipients (6) The literature docs not refer to research into the kinetics of MHC in substance in solid phase, ‘The aim of the study was to evaluate the influence of relative humidity and temperature on the stabili- ty of moexepril hydrochloride during storage of the substance EXPERIMENTAL Materials and reagents Moexipril hydrochloride was obtained from Schwarz-Pharma AG. Other chemical substan o1 cces and reagents were products of Sigma Chemi: cal Co, HPLC method for the estimation stability of MHI in solid phase In this paper, a modified HPLC method was applied, used earlier for the study of stability of enalapril maleate and quinapril hydrochloride in solid phase (7.8) ‘A Merck analytical column (Hypersil MOS, 5 jm particle size, 250 mim x 4 mm id.) was used as the stationary phase. The mobile phase: acetonitrile ~ phosphate buffer (pH = 2.0, 0,001 molelt} (5 viv}; flow rate of the mobile phase: 1.3. mV/min; internal standard: xylometazoline hydrochloride (met hhanolie solution 0.16 mg/m): detector UV: 220 nm. HPLC ~ MS method for the i decomposition product of MHCL Column: Hypersil MOS, 5 jum particle size. 250 mm x4 mm i.d. Merck: mobile phase: acetoni- tile ~ water (90:10) with formic acids, flow rate: 0.5 ml/min, lentification of Validation method For the validation test, the following substan- fees were used: a sample of MHCI, a sample of MHC heated to 373 K in dry air, and a sample of MHC heated to 363 K in a humid atmosphere of 76.4% RH. During the validation siudy, the fol- lowing parameters were evaluated: selectivity: the applied method was selective for the MHCI ( = about 5 min) as well as for the92. BEATA STANISZ internat standard (xylomtetazotine hydroehforite 1 = about 7 min) in the presence of degradation products (tk = about 2 and 3 min); Precision: it was were evaluated for 10 individu- af samples of 0.4 mg/ml MHCI, and the following results were obtained: mean value P/P,, = 2.3115, standard deviation = 0.0248, variation coefficient 075%: Tinearity: the valibration curve for MHC was taken in methanol for solutions of concentrations varying from 0.0125 mg/ml to 0.6000. mg/ml. ‘The imernal standard (xylometazoine hydro- chloride) was a methanolic solution at « concent- ration of 0.16 mg/ml. Next, 1.0 ml of the so obtained solution was mixed with 1.0 ml of the internal standard solution and subjected to analy’ sis, Linearity was also examined for three succe- ‘eding days in solutions of the same concentration prepared from a stock solution. The equation for the calibration curve was: ¥ = (SAS + 0.14) x 6; for the equation y = ae + b, the value b was statistically insignificant). The calculated cor relation coefficient was > 0.999, thus indicating 00d tiaearity: limit of detection (LOD), limit of quantification (LOQ): in the described experimental conditions the limit of detection was approx. 6 g/ml and the it of quantification was 12 jig/ml: repeatability of the method was tested by eight replicates and evaluated by the variation coefficient 31%. Conditions of the Kinetic studies For the experiments, 10 mg samples of MHCI were weighed into 5 ml open vials, To assess the effect of humidity on the stability of MHCI, the vials with MHCI were placed in desiccators containing aqueous saturated solu- tions of appropriate inorganic salts, which safe Buarded the conditioned relative humidity: so- dium bromide (RH = 50.9%), potassium iodide (RH = 60.5%), sodium nitrate (RH = 66.5%) and soxtiua clilorigde (RH = 76.4%) and inserted in a heat chamber set to 363 K. Samples destined for investigation of the ef- {ect of temperature at a relative humidity of 76.4% were placed in desiceators containing aqueous satt- ‘ated solutions of sodium chloride and inserted in hheat chambers set up to the desired temperatures: 333 K, 343 K, 353 K, 358 K and 363 K. To assess the stability of MHICI in dry air, the vials containing the studied substance were i sed in a sand bath, in a heat chamber adjusted to 4 temperature of 353 K. 358 K, 363 K, 373 K and 383K. Doring the experiment, the color of the sub- stance changed from white through yellow 10 brown, all produets of degradation were soluble in ‘methanol. Each series comprised 7 ~ 12 samples. ‘After definite time intervals, determined by the rate of degradation, the respective vials were taken out of the chamber, cooled (© room tem perature and the conienis dissolved in methanol The so obtained solution was quantitatively tan ferred into a measuring flask and made up to the total volume of 25.0 ml with methanol. To 1.0 ml Df the so obtained solution 2.0 el af the iacennat standard solution was added. The internal standard |was added to the Solutions at a constant concent- ration before investigation 100 4 of the samples were fed to the HPLC column and the emerging signals were recorded. “The chromatograms were interpreted using the following dependence: P/P,, = ft; where P, are the values of the MHCl, and P,, represent the values of internal standard, ¢ represent time, RESULTS AND DISCUSSION Rate constants of degradation reaction of MHCI In presence of relative humidity (30.9% ~ 164%), the changes in concentration of MHC ‘occurred according 10 the first onder reaction mo- Wel, ko > 0, The plots In c = ig) were linear (Figures 1 and 2) and the observed rate constants were calculated hy the Jeast squares method accor- ding to the equation: Inc = In cy — kt, where: ¢; and co represent the concentration of MHC in time 1 and 0, respectively, & is the Birst-order rate constant (5, ‘The following statistical parameters of the respective equations were computed: a + Aa, 330K so 400 ous Figure 1, Sengacithn plots foe the degra f MCL in soft pse at reine hmishy RAL TO at diferet tom pears,‘The intuence of relative humidity and terperaice on sbi Table I, Kinetic and theanadynaricparanters of MEIC dsimpostion in sli phe at relatve hum 93 Tk) wera | or) ow Statistical evalution “Thennodsaie | | Ink = fT) parameters Relative humidity, RH = 76.4% ] 16.96 + 7.9 333 | 0106 £008 0998 10 | at da =-14067 2 044 (Wrote MB | 03% F002 | 097 | 7 | 5,= 2909 ana nasi +78 31205 F014 | 099 | 7) hEAD= 285427 thamotr ass | i9szoar | 96 7 | sys oss as = 829-4 224 [38 samt 099 9 | pe -o959 100K mol | Dry alr, RH = 0% | Be 1453 £22 383 | o1oszo0%9 | 098 | wo | at astm £29 {km} 358 0218 £0022 | O97 7 83 an = 1428 225 36302934014 | 095 | 7 | bean = A574 745 (hamote Bs tas £021 | 0.996 | 7) = 269 ast= S172 188 ws | somreoat | os | 9 ogy LAM % mole) 100 ~ —— + . i 6%) | . 50.9% | 10 . i . 605% | t ' th | 04 — ! ° 200 ° hours. 50 100 gues 150 Fgwe 2, Semigarthimie pts or the deyraton f MHC in 10° ee ‘oh phase a teeta emp. 8 Ke 9 od ee, ees . ae “eo SK b+ Ab, standard derivative (SD) and the coefficient 1/2 Bo : of lincar correlation. The values of Aa and Ab were 3; Oy 38K 73K K ‘computed for f = n-2 degrees of freedom, with al? a e005. i In dry air, the concentration changes of MHCL | Ase3 k occurred according to the pseudo-first-order reac: 901 ~ tion model; ¢; > ¢,50 (Figure 3a). The plots In ° 20 400800 ors 80 (cec,) = ff) were linear (Figure 3b) and the rate constants. were calculated by the least squares method aeconding 10 the equation: Im (6, 6.) = In (en) —kot ‘where: ¢, ¢ and ¢, represent the concentrations of ‘MHC in time ¢, de infinite and 0, respectively, k is the first-order rate constant (s") Figure 3a. Changes of MIC convention during storage a RH Vial 3 Ke 1. Semoun plas Ine, e,) = fe the degrtion ot IIHCT i oll pine indy sea tere temperate, For the interpretation of the straight curves plotted from tn (c, — ¢) = {00 the following Matistical parameters of the respective equations4 ‘were compuled 6y means oF the minimal squares methods a + Aa, b+ Ab, standard derivative (SD) and the coetTicient of linear correlation, The values fof Aa and Ab were computed forf = n-? degrees of freedom, with @ = 0.05. ‘Thermodynamic parameters ‘The determined reaction rate constams were employed for the calculation of the Atrhenias ‘elationship: Ik, = Jud — E/RT, where k; represent the respective reaction rate constants [5-"}.A quency coefficient, £, = activation energy U/mole], BEATA STANISZ. 1.00608 1 | | 106.05 . 1100-06 0 om mR, gp Fie 4 Te effec of humy onthe stability of MICH soit hose Product of hydrolysis of MHCI (m/z = 470) ae 13204 1: Sean s+ “oo, a % 2 208 re m 099 Zn sos 28 8 aos [i Pese 9 H8 ore 7% 150 Pour 1? oye S48 200" “360 abo 550 650" F000 GO FOO 3.293 2: Sean ES- “9 4 28 25 yg sez Eagif? 150 505 £71 aug 7a 779 agg R40 HRI oh i 200" gaa 400" "S80 "80" 780™” OBO TOC Product of intramolecular cyclization of MHCI (m/z = 480) 068 1: Scan Est Figure 5. ass spectra eration rods of MCL‘The inoence of relive humiiy and temperature on tabi 95 “Table 2, The effet of humidity on the ity of MHICL in slid phase t 363 K ane | kt ak se | | Parameters of regression is Ink | 50.9 0,578 + 0.030 0.991 8 | | os 1.085 + 0.301 09% | 10 eos | 1aset0303 | 091 | 7 roa | s30s0308 | os | 9 ore e000%m Meare meeGan wel DATO co % gooey M480 Scheme 2. Depraistion of MHC in sf phase (experiment canions: RM = 0%, temp. 373K), R = universal gas constant (8.3144 I(K x mole), temperature [K_. For the relationship in &, = /lU/T) straight line plots were obtained for both the humid and dry conditions of sample exposure (Table 1). From the parameters of the plot in k = f(I/T) the following thermodynamic parameters of the de composition reaction of MHCI in solid phase, pertaining to either conditions of sample incuba- ti mn, ie. im dry air and in an atmosphere of RH 76.4% were calculated: the activation energy (E,). enthalpy (AH’) and entropy (AS*) for the temperature 293 K, Effect of humidity on the stability of MHCL The effect of humidity on the stability of MHC at 363 K, in the humidity range from 50.05% to 76.4%, is described by the equation: In k, = ax + b= (0.0676 + 0.0163) x RH% ~ (15.53 * 0.775) (Figure 4). The slope (a = 020676 + 0.0163) of the straight linear plot in ky = (RH%) characterizes the effect of humidity on the stability of MHCL ‘This effect is similar to the effect of humidity on the stability of quinaprit hydrochloride (a = 0.0589 £0.0086) (8), The value b obtained by extrapolation of this dependence for RH = 0% (b= ky = 1.802 +1075") is lower than the value kat 363 K in dry air ‘conditions appointed empirically (k = 2936 = 10°", Such results poiot tothe differences in the shanism of degradation of MHCI occurring in the presence or absence of ambient humidity (Table 2) Identification of the degradation products. of ‘MHCI (relative humidity 76% and temp. 363 K) In the presence of relative humidity, the fol- owing signals were observed on HPLC-MS chro-96 BEATA STANISZ -matogram: the MHC! signal at ty = about 25 min, ‘and two signals corresponding 10 the decomposi- tion produets of MHCI ty = about 5 and 13 min. Mass spectra of the degradation product of MHCT obtained by meaus of the technique of ESI alfowed to define their molecular masses M = 470. mlz = 471; ES mlz about 5 min; ES*, miz = 481). The analysis of HPLC-MS spectra yielded the following coaclusion: the product with M = 470 is mocxipril diacid and the product with M=480 is moexiprl diketopiperazine (Scheme 1, Figure 5) Adentifcation of the degradation products of MHC in dry air (RH = 0%, temp. 373 K) Tn dry ar, the following signals were observed ‘on HPLC-MS chromatogram: the MHCI signal at te = about 25 min, and one signal comresponding to the decomposition product of MHCI ty = about 5 min. ‘Mass spectra of the degradation product of MHC obtained by means of the technique of ESI allowed to define their mofecufar masses M = 480 Gq = about S min; ES*, my = 481). The analysis of HPLC-MS spectra yielded the following conclu- sion: the product with M = 480 is moexipril diketopiperazine (Scheme 2, Figure 5). CONCLUSIONS ‘The HPLC method was found to be selective, linear, accurate and precise for the evaksation of stability of MHCT in solid phase. In dry atmos- phere (RH = 0%, in the temperature range from 343 K to 383 K), only moexiprl diketopiperazine was the product of degradation of MHC1in solid phase. In the presence of humidity (RH from 50.0% to 76.4%, in the temperature range from 333 10 363 K) moexipril diacid (maexiprifat) and) moexipril ciketopiperazine were the products of degradation ‘of MHCI in solid phase. The conducted study on the stability of MHCI has shown that the inves- tigated substance (in solid state) presenis 2 ap- preciable siabibiy at room temperature, The de tradation constants of MHCI at 298 K when extrapolated from the Arthenius equation, are equal to k= 1.005 «10! 8 fun = 12134 days: to = 79783 days (RH = 0%) and k= 7.012 10° 143 days (RH = 766), Acknowlegements I wish to thank the company SCHWARZ PHARMA AG for supplying moexiprihydrochlo: ride pace substance. 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