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**Journal of Pharmaceutical and Biomedical Analysis
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Short-term stability studies of ampicillin and cephalexin in aqueous solution and human plasma: Application of least squares method in Arrhenius equation

Ticiano Gomes do Nascimento a,∗ , Eduardo de Jesus Oliveira b , Irinaldo Diniz Basílio Júnior a , João Xavier de Araújo-Júnior a , Rui Oliveira Macêdo c

a Laboratório de Controle de Qualidade de Fármacos e Medicamentos, Curso de Farmácia, Escola de Enfermagem e Farmácia, Universidade Federal de Alagoas, Campus A. C. Simões, BR 104 Norte, Km 97, Maceió, AL CEP:57072-970, Brazil b Programa de Pós-Graduac ¸ ão em Produtos Naturais e Sintéticos Bioativos, Centro de Biotecnologia, Universidade Federal da Paraíba, Campus I, CEP: 58051-970, João Pessoa, PB, Brazil c Laboratório de Controle de Qualidade de Produtos Farmacêuticos, Departamento de Ciências Farmacêuticas, Universidade Federal de Paraíba, João Pessoa, PB CEP:58059-900, Brazil

a r t i c l e

i n f o

a b s t r a c t

A limited number of studies with application of the Arrhenius equation have been reported to drugs and biopharmaceuticals in biological ﬂuids at frozen temperatures. This paper describes stability studies of ampicillin and cephalexin in aqueous solution and human plasma applying the Arrhenius law for determination of adequate temperature and time of storage of these drugs using appropriate statistical analysis. Stability studies of the beta-lactams in human plasma were conducted at temperatures of 20 ◦ C, 2 ◦ C, −20 ◦ C and also during four cycles of freeze-thawing. Chromatographic separation was achieved using a Shimpak C18 column, acetonitrile as organic modiﬁer and detection at 215 nm. LC-UV–MS/MS was used to demonstrate the conversion of ampicillin into two diastereomeric forms of ampicilloic acid. Stability studies demonstrated degradation greater than 10% for ampicillin in human plasma at 20 ◦ C, 2 ◦ C and −20 ◦ C after 15 h, 2.7 days, 11 days and for cephalexin at the same temperatures after 14 h, 3.4 days and 19 days, respectively, and after the fourth cycle of freezing–thawing. The Arrhenius plot showed good prediction for the ideal temperature and time of storage for ampicillin (52 days) and cephalexin (151 days) at a temperature of −40 ◦ C, but statistical analysis (least squares method) must be applied to avoid incorrect extrapolations and estimated values out uncertainty limits. © 2012 Elsevier B.V. All rights reserved.

Article history: Received 8 December 2011 Received in revised form 6 April 2012 Accepted 9 April 2012 Available online 20 April 2012 Keywords: -Lactams Human plasma Arrhenius equation Stability prediction LC-DAD LC-UV–MS/MS

1. Introduction Penicillins and cephalosporins antibiotics have been the most widely used antimicrobial drugs at long of the years and they are still considered as one of the most important groups of antibiotics [1]. Despite their extensive and long standing use in therapeutic, there has been incomplete description of the stability of drugs, specially the beta-lactams antibiotics in biological ﬂuids. Other therapeutic classes of drugs are naturally unstable in bioﬂuids and include: acetylsalicylic acid [2], thiolic derivatives [3], Zopiclone [4], antimalarial drugs [5] and benzodiazepines [6]. Stability study in bioﬂuids is an important prerequisite for validation of a bioanalytical method [7] and has practical implications in the drug determination during pharmacokinetic evaluation [8] and can avoid errors in bioanalytical laboratory by assuring a more effective patient management, including diagnosis,

∗ Corresponding author. Tel.: +55 021 82 3214 1155; fax: +55 021 82 3214 1155. E-mail addresses: ticianogn@yahoo.com.br (T.G. do Nascimento), eoliveira@gmail.com (E. de Jesus Oliveira), ruiomacedo@yahoo.com.br (R.O. Macêdo). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.04.010

treatment of disease, clinical monitoring and disease prevention [9]. Stability studies have a great relevance in biological systems to evaluate the chemical integrity of drugs and biopharmaceuticals and the determination of the kinetic of degradation of analytes including monitoring of degradation product using LC-tandem MS [10]. Stability studies and classical Arrhenius method have been scarcely used in biopharmaceutical analysis to assess the chemical stability, potency, purity, quality and shelf life of drugs and biotechnological products [11] under isothermal or non-isothermal conditions [12], mainly at the frozen temperatures. Statistical analysis was applied to evaluate the data uncertainty using linear or non-linear methods [13]. Application of the short-term stability studies can be an alternative to reduce the cost of the bioanalytical validation program and to evaluate the behavior of drugs in biological matrices. This article presents some analytical data of beta-lactams in stock solution and human plasma at different temperatures during short-term stability studies. The purpose of this study was to apply the Arrhenius equation in the stability study of ampicillin and cephalexin in order to determine adequate temperature and time of storage of these drugs using an appropriate statistical analysis.

2. Stock solution Ampicillin and cephalexin (50 mg) were weighed and quantitatively transferred to a 10 mL volumetric ﬂask to obtain a (5 mg/mL) stock solution in ultrapure water. which were obtained according to graphical analysis using Arrhenius equation (4) and the time of storage was also calculated [15]. The LC-UV–MS/MS conditions for detection of the beta-lactams included a mobile phase that consisted of acetonitrile: formic acid (0. v/v) delivered at a ﬂow rate of 1. Results and discussion 3. 2. i. potassium dihydrogen phosphate and HPLC-grade acetonitrile were purchased from Merck. Micromass) was used for determination of ampicillin. i. An upper and lower limit of 10% of the original concentration was established as acceptance criteria for stability. an autosampler SIL-10AD. 0. All other reagents were analytical grade. 2.0 mL. The pH of the phosphate buffer was adjusted with 4% phosphoric acid. Stability studies in stock solution and human plasma Aliquots of 1. It was operated in positive electrospray ionization mode with a capillary voltage of 2.2. 2. respectively. R = gas constant. The mobile phase consisted of acetonitrile: dihydrogen phosphate (pH 3.5. The degradation rate constant (k) at sub-zero temperature was estimated by extrapolation of the best linear regression with least squares method for the temperature of −30 ◦ C and −40 ◦ C. Perchloric acid. Application of Arrhenius equation and statistical analysis The HPLC system consisted of an LC-10AD pump.5. Germany. k0 = zero-order degradation rate constant.d. named CQ1 .6 mm. Brazil) after approval from the research ethics committee.1.G. 2 ◦ C and −20 ◦ C using Arrhenius equation as time function (Eqs. Separation was achieved at 25 ◦ C using a Shimpak C18 column (250 mm × 4.d 5 m) ﬁtted with Shimpak security guard C18 column (4.5.0 mL min−1 that was reduced with a splitter to 0.3. A = preexponential factor. Two stock solutions containing only one analyte were submitted to aging for 10 days at 20 ◦ C and analyzed. and a controller module SCL-10A (all from Shimadzu) coupled to a personal computer running the software Shimadzu Class VP for data acquisition.0.5 and 6.0 mm × 3.5: 87. 2.2 mL min−1 for introduction into the mass spectrometer. QC2 and QC3 ) was used to calculate the order of reaction and degradation rate constant (k) in each temperature studied 20 ◦ C.0 mm.0 mm. The ﬂow rate was set at 1.70 kV. i. Ea = energy of activation. Samples were introduced using the autosampler and the injection volume was 40 L. k2 = second-order degradation rate constant. Non-spiked fresh plasma samples were also stored in the same conditions and at the moment of analysis were spiked with analytes in order to obtain in all the samples the same nominal concentration of the aged samples and calibration curve samples. Darmstadt. Paraíba. All analyses were done in triplicate. These peaks were denominated AMP A1 and AMP A2. . do Nascimento et al. 1 and 4A and B).1 min became apparent in the period after 15 days of storage. Experimental 2.0 g/mL) in stock solution or human plasma. Additional experiments were done to determine the origin of the degradation products.4. It was calculated the coefﬁcient of correlation (r2 ).5. 10. The 95% conﬁdence interval and 95% prediction interval were used to obtain the upper and lower limits of variations [16].0 mL min−1 and a discrete channel on the diode array detector conﬁgured to acquire data at 215 nm. (1)–(3)) [15]. Mexico) and cephalexin pharmacopeial standard (from Brazilian Pharmacopeia) were used throughout the study. i. Full scan acquisition was made in the range of 40–400 m/z with the multiplier voltage at 650 V. degradation products (AMP A1 + AMP A2) were formed uniquely from ampicillin after 10 days of storage at 20 ◦ C (Figs. In each temperature. 5 m). 5 m) ﬁtted with a Phenomenex security guard C18 column (4.0 mm × 3.1 M) (12. the mean of the degradation rate constant (k) (from triplicate experiments) was obtained and did not present variation greater than 15% of relative standard deviation (RSD). Human plasma samples Human plasma (250 mL) collected from healthy volunteers and stored in bags containing heparin as anticoagulant was acquired from the Hemotherapy Center of Paraíba (João Pessoa.3.1.0 and 50. Successive dilutions were made to prepare quality control samples at concentrations of 1.2.d. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 59–64 2. tx = ﬁnal time. Stability of beta-lactams was evaluated by comparing the responses of ampicillin and cephalexin in stored samples and with those freshly prepared sample at the same nominal concentration level. a diode array detector (SPDM10A). Sample preparation 2. Separation was achieved using a Shimpak C18 Column (250 mm × 4. The method for quantiﬁcation of ampicillin and cephalexin during the stability studies has been previously validated [14]. sampling cone voltage of 21 V. Reagents and chemicals Ampicillin USP (from Ferjinsá. LC-DAD and LC-UV–MS/MS conditions CQ2 and CQ3 .3.0.0 and 50. t0 = initial time. residual analysis and estimating the standard error (SYX ) in the Arrhenius plot (ln k × 1/T) using the least squares method. desolvation gas temperature of 250 ◦ C (N2 ) and source block temperature of 110 ◦ C. Brazil).1%) (10:90. 2 ◦ C and −20 ◦ C during 15 days. A triple quadrupole mass spectrometer (Quattro LC. Each quality control sample (QC1 . a column oven (model CTO-10AS).1. were transferred into cryovials and stored at 20 ◦ C. to promote sample aging. Cx = concentration of the drug in ﬁnal time. The stock solutions were stored in cryovials of 2. k0 = C0 − Cx tx − t0 Cx /C0 tx − t0 (1) (2) (3) (4) k1 = ln k2 = (1/Cx ) − (1/C0 ) tx − t0 A − Ea RT ln k = ln where C0 = initial concentration. Water was puriﬁed with a reverse osmosis system connected to an ion exchange unit (Gehaka. phosphoric acid. 2. v/v). 3. Degradation products appeared in the ampicillin stock solution (10 g/mL) but not in cephalexin stock solution (10 g/mL). T = absolute temperature in Kelvin.0.0 mL from the quality control samples (5. Stability studies in stock solution During the stability study in stock solution the appearance of two new chromatographic peaks at 5. its related degradation products and cephalexin. 10. Thus. 30 days and 103 days.0 g/mL.6 mm. 5 m) kept at 40 ◦ C in a column oven.d. 5. extraction cone voltage of 3 V. k1 = ﬁrst-order degradation rate constant.60 T. Analysis of variance (ANOVA-one way) and P-value were used to check if the experimental values followed the Arrhenius plot.

T. 3). The mass spectra of both AMP A1 and AMP A2 presented a mass gain of 18 amu in relation to ampicillin. Chromatograms in MS/MS detection (A) and (B) in scan mode (50–400 m/z) and chromatogram in UV detection (C).5 and 15. The conversion of the ampicillin in ampicilloic acid 1 (5S.0. 6R) in aqueous medium. . 6R) exhibited a lower retention time than its (5R. 14. Fig. AMP A1 (5S. The detection of ions at m/z 368 with retention times 8. The LC-UV chromatogram showed presence of AMP A1.0 (AMP A1) and 9. 9. Stock solution containing ampicillin and cephalexin aged during 15 days at 20 ◦ C were analyzed using full scan mode. which suggests a hydrolytic reaction involving ampicillin and a water molecule. do Nascimento et al.0 min (AMP Fig. 2). 6R) diastereoisomer and our data have corroborated these results [18]. 6R) and 2 (5R. AMP A2. The spectra also presented an uncommon ion (at m/z 324) that is related to the loss of carbon dioxide (CO2 ) and suggests the presence of a second carboxylic acid group in the molecule. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 59–64 61 Fig. 6R) and 2 (5R. The extracted ion chromatograms displayed two peaks at m/z 368 with retention times of 8. in agreement with a previously validated method [14]. [17] reported several degradation products from ampicillin including epimeric forms of ampicilloic acid. ampicillin and cephalexin at retention times of 8. of the ampicilloic acid from the ampicillin degradation.0. It is reported that degradation of cephalexin to 7-aminodesacetoxycephalosporanic acid is usually found when the drug is subjected to extreme conditions such as acidic or alkaline conditions (pH 1 or pH 8) and in this conditions cephalexin is 180 times more stable than ampicillin [19]. 6R) in acid aqueous medium can be demonstrated during sample preparation of -lactams in stock solution. respectively. LC-UV–MS/MS experiments were done in order to identify the degradation products (AMP A1 and AMP A2) (Fig. Chromatographic conditions acetonitrile:formic acid (0. 2. 3. 6R) and AMP A2 (5R. (C) Chromatogram of separated stock solution of ampicillin (10 g/mL) and (2) cephalexin (10 g/mL) after 10 days of storage at 20 ◦ C. A2) in the extracted ion chromatograms and the mass spectral data supported this evidence and has conﬁrmed the formation of two epimeric forms. in which the ampicilloic acid (5S. LC-UV–MS/MS chromatogram of ampicillin (10 g/mL) and cephalexin (10 g/mL) with detection of diastereomeric forms of ampicilloic acids (AMP A1 and AMP A2) in stock solution. (B) Chromatogram of stock solution (1) of ampicillin (10 g/mL) and (2) cephalexin (10 g/mL) after 15 days of storage at 20 ◦ C with detection of two degradation products (AMP A1 and AMP A2). v/v).1%) (10:90. 1 (5S. 1. The scan mode (using collision-induced dissociation) allowed obtaining more information about the fragmentation pattern of the ions of ampicillin (m/z 350) and theirs degradation products (m/z 368). (A) Chromatogram of drug-free plasma (a) and spiked plasma (b) with (1) ampicillin (10 g/mL) and (2) cephalexin (10 g/mL). Scheme illustrating the ampicillin conversion in two diastereoisomeric forms of the ampicilloic acid.4 min. Zhu et al. 6R). The opening of the -lactamic ring by acid hydrolysis is the accepted mechanism for formation of these two ampicilloic acid diastereoisomers (Fig.0 min and a peak with m/z 350 at the retention time corresponding to ampicillin.G.0 and 9.

a ﬁrst-order kinetic for both beta-lactams. 3. During the stability study. The study of freeze–thaw cycles also revealed a high variability of the measurements (>15%) after the third cycle and lower recovery of ampicillin (84. 3. 60 days at 2 ◦ C. conditions for cephalexin quantiﬁcation are more restrictive than ampicillin. Quantitative loss mass of ampicillin (A) with simultaneous conversion in two degradation products AMP A1 and AMP A2 in stock solution. using the (k1 ) ﬁrst order degradation constant (Table 1). the formation of interferents from the plasma aging process (30 days at room temperature. Thus.62 T. and 103 days at −20 ◦ C) resulted in coelution with cephalexin. The ideal time of storage involving processing and analysis of extracted samples is shown in Table 1. At 24 h of storage ampicillin and cephalexin presented degradation greater than 10% (Fig. Stability studies of ampicillin and cephalexin in human plasma (C) storage at room temperature. whereas cephalexin was stable for 20 days within limits established (<10%) (Fig. 4C–E).2. 4C). Estimative of 95% conﬁdence interval (solid line) and 95% prediction interval (dashed line) for the extrapolation up to the temperature of −40 ◦ C of the degradation rate constant (k1 ) of ampicillin (G) and cephalexin (H) using Arrhenius plot. (D) storage at 2 ◦ C.3. At freezer temperature (−20 ◦ C) the degradation of ampicillin is delayed for 10 days. Stability studies in human plasma The stability study of ampicillin and cephalexin spiked in human plasma revealed degradation of the drugs after 6 h of storage at 20 ◦ C. Ampicillin and cephalexin spiked in human plasma presented degradation levels close to 40% after 15 days in the stability study at 2 ◦ C (Fig. Application of Arrhenius equation and statistical analysis The data showed a similar kinetic of degradation at 20 ◦ C (second-order kinetic) and at low temperatures.7%) and cephalexin (88. The study of freezing and thawing cycles showed that the ampicillin and cephalexin were stable for three consecutive cycles within limits (<10%). 4. (E) storage at −20 ◦ C and (F) after freeze–thaw cycles.G. (B) quantitative loss mass of cephalexin in stock solution. 2 ◦ C and −20 ◦ C. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 59–64 Fig. The k1 values were extrapoled applying the least squares method into the Arrhenius plot ln k × (1/T) with the best ﬁt observed by coefﬁcient of . ampicillin and cephalexin.9%) which was observed in the fourth cycle of freezing and thawing (Fig. do Nascimento et al. 4D). The degradation rate constant of the ampicillin and cephalexin were assessed as time function (Fig. 4F). It was calculated the extrapolated degradation rate constants (k1 ) values at sub-zero temperature and the maximum time of storage to the analytes. 4E). Arrhenius plot was applied to assure a more long time of storage for beta-lactamics in human plasma and to avoid degradation of drugs during the analysis.

6%.6%) and cephalexin (5.9978 0.8 r2 0.3x ± 177 0. but statistical tools must be employed to avoid incorrect extrapolations and estimated values out uncertainty limits.015 0.982 0.989 0.0016 0.7 10.000744 Time (days) 50.000006 Cephalexin k1 0.4 19. Ampicillin and cephalexin could be preserved for longer periods of storage at sub-zero temperature (−30 ◦ C and −40 ◦ C). Substitution of the mean values of rate constant (k1 ) in each temperature by individual values of rate constants (k1 ) obtained for each quality control samples (CQ1.1173 −8. t2(90) = 0.14 0.58 3.713 1066 0. k0 = (C0 − Cx )/(tx − t0 ). correlation and residual analysis.000032 0.0013 0.8%) and cephalexin (20.004468 0.0312 ± 0. 4G and H has demonstrated good estimative of standard error (SYX ) for ampicillin (8.00171a 0.81 ± 0.06788 −7.219 ± 0.002068 0.79 ± 1.878 Each value is expressed as mean ± SD of three determinations of the QC samples. when submitted to natural processes of degradation in aqueous medium or biological conditions have followed the classical Arrhenius method. t1(90) = 0.974 0. Estimative of conﬁdence interval of degradation rate constant (k1 ) was extrapolated for temperatures of −50 ◦ C.993 0. Conclusion Ampicillin and cephalexin presented instability in human plasma after short periods of storage at temperature of −20 ◦ C. which was calculated to determine the experimental error of the best linear curve for ampicillin (6. 4G and H showed the best regression curve with application of the estimative of conﬁdence interval for the extrapolation calculi of the (k1 ) values of ampicillin and cephalexin at sub-zero temperature (−40 ◦ C).87 2.0195 0.00157 ± 0. do Nascimento et al.013 to −4.000061 ± 0. as demonstrated in Table 1 and thus adequate in the previsibility of the extrapolated time of storage.0090 0. b k1 = ﬁrst-order degradation rate constant.0025 0.956 0.00005 0.000523 ± 0.0381b 0.910 First order k1 0.0297 Cephalexin Linear regression r2 SYX ln k (−40 ◦ C) 95%CI of ln k(−40 ◦ C) F P < 0.0054b Time (days) 0.0097b Time (days) 0. while cephalexin would be preserved for 52 and 141 days.000432 ± 0.5 52.966 0. The extrapoled degradation rate constants (k1 ) values of ampicillin and cephalexin presented relative standard deviation (RSD) of 2.1237 ± 0. a k2 = second-order degradation rate constant.0024 0. which would be sufﬁcient to preserve ampicillin for periods of between 20 and 50 days. 4.105/k1 .0381 ± 0. linear methods have some advantages over the non-linear methods because they are robust to deviations from homoscedasticity [13]. Ampicillin presented greater variation in (SYX ) and thus resulting in major amplitude in the hyperbolic effect of the conﬁdence bands in relation to cephalexin.0097 ± 0.983 0.7 0.9991 0.001991 Time (days) 23. The planning of this stability studies only in three different temperatures was not sufﬁcient to minimize the amplitude of the hyperbolic effect of conﬁdence bands. but there was no success in the calculi [16]. The minimum sample size of (k1 ) and the estimative of standard error (SYX ) contributed for the large conﬁdence interval of the ln k (−40 ◦ C) for ampicillin (28.8%).3% and 1.0312b 0.425 469. ampicillin and cephalexin.32 ± 0.387 − 4568.0054 ± 0.8 141.1x ± 213 0.0500 y = 17.987 0.204 ± 0.2%) and cephalexin (6.2 y = 13.990 0.000109 ± 0. (◦ C) Zero order k0 Ampicillin 20 2 −20 Cephalexin 20 2 −20 Temp. degradation rate constants and estimative of storage time at sub-zero temperature for ampicillin and cephalexin applying the least squares method in Arrhenius equation. The beta-lactams.609 − 5781. (◦ C) −30 −40 k1 b 0. (◦ C) −30 −40 k1 b 0.18 8.00157a 0. The best regression curve presented in Fig.991 0.39 2.77 ± 0.88 ± 0. Temp.00171 ± 0.T.7%) (Table 1).962 0. .953 0.0002 r2 0. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 59–64 63 Table 1 Determination of order of reaction. In some cases.4 r2 0. respectively. k2 = [(1/Cx ) − (1/C0 )]/(tx − t0 ). The analysis of variance (F) and P value demonstrated signiﬁcant difference in relation to the values (k1 ). Fig.1412 −8. (◦ C) 8.987 0. The Arrhenius equation was useful to evaluate the degradation kinetic of beta-lactams and estimate the ideal time and temperature of storage.64 2.0500 Prediction of Arrhenius parameters Ampicillin Temp.G.50 0.00005 0.993 0.7 Cephalexin Temp.00027 0. CQ2 and CQ3) has reduced the hyperbolic amplitude on the regression analysis [16].001/k2 . k1 = ln[Cx /C0 ]/(tx − t0 ).42 ± 0.695 to −5. Based on this special class of drugs very unstable in biological ﬂuids it was considered the propose to determinate the adequate time and temperature of storage in short-term stability studies during the step of method validation.5%).899 Second order k2 0.1157 ± 0. thus the degradation rate constants values obeys the Arrhenius law and were dependent on the storage temperature [15] (Table 1). respectively.12 Ampicillin k1 20 2 −20 Statistical parameters Ampicillin Linear regression r2 SYX ln k (−40 ◦ C) 95%CI of ln k(−40 ◦ C) F P < 0.08169 −6.995 0.

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