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 ARTICLE IN PRESS
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Journal of Pharmaceutical and Biomedical Analysis xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

1 Short communication

2 Short-term stability studies of ampicillin and cephalexin in aqueous solution and

3 human plasma: Application of least squares method in Arrhenius equation
4 Q1 Ticiano Gomes do Nascimento a,∗ , Eduardo de Jesus Oliveira b , Irinaldo Diniz Basílio Júnior a ,
5 João Xavier de Araújo-Júnior a , Rui Oliveira Macêdo c
6 Q2 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,
7 BR 104 Norte, Km 97, Maceió, AL CEP:57072-970, Brazil
b
8 Centro de Biotecnologia Prof. Delby Fernandes de Medeiros, Universidade Federal de Paraíba, João Pessoa, PB CEP:58059-900, Brazil
c
9 Laboratório de Controle de Qualidade de Produtos Farmacêuticos, Departamento de Ciências Farmacêuticas, Universidade Federal de Paraíba, João Pessoa, PB CEP:58059-900, Brazil
10

11 a r t i c l e i n f o a b s t r a c t
12
13 Article history: A limited number of researches with application of the Arrhenius equation have been reported to drugs
14 Received 8 December 2011 and biopharmaceuticals in biological fluids at frozen temperatures. This paper describes stability studies
15 Received in revised form 6 April 2012 of ampicillin and cephalexin in aqueous solution and human plasma applying the Arrhenius law for
16 Accepted 9 April 2012
determination of adequate temperature and time of storage of these drugs using appropriate statistical
Available online xxx
analysis. Stability studies of the beta-lactams in human plasma were conducted at temperatures of 20 ◦ C,
17
2 ◦ C, −20 ◦ C and also during four cycles of freeze-thawing. Chromatographic separation was achieved
18 Keywords:
using a Shimpak C18 column, acetonitrile as organic modifier and detection at 215 nm. LC-UV–MS/MS
19 ␤-Lactams
20 Q3 Human plasma was used to demonstrate the conversion of ampicillin into two diastereomeric forms of ampicilloic acid.
21 Arrhenius equation Stability studies demonstrated degradation greater than 10% for ampicillin in human plasma at 20 ◦ C, 2 ◦ C
22 Stability prediction and −20 ◦ C after 15 h, 2.7 days, 11 days and for cephalexin at the same temperatures after 14 h, 3.4 days
23 LC-DAD and 19 days, respectively, and after the fourth cycle of freezing–thawing. The Arrhenius plot showed
24 LC-UV–MS/MS 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.

25 1. Introduction diagnosis, treatment of disease, clinical monitoring and disease pre- 40

vention [9]. 41

26 Penicillins and cephalosporins antibiotics have been the most Stability studies have a great relevance in biological systems 42

27 widely used antimicrobial drugs at long of the years and they are to evaluate the chemical integrity of drugs and biopharmaceuti- 43

28 still considered as one of the most important groups of antibiotics cals and the determination of the kinetic of degradation of analytes 44

29 [1]. Despite their extensive and long standing use in therapeutic, including monitoring of degradation product using LC-tandem MS 45

30 there has been incomplete description of the stability of drugs, [10]. Stability studies and classical Arrhenius method have been 46

31 in specially the beta-lactams antibiotics in biological fluids. Other scarcely used in biopharmaceutical analysis to assess the chem- 47

32 therapeutic classes of drugs are naturally unstable in biofluids and ical stability, potency, purity, quality and shelf life of drugs and 48

33 can be including: acetylsalicylic acid [2], thiolic derivatives [3], biotechnological products [11] under isothermal or non-isothermal 49

34 Zopiclone [4], antimalarial drugs [5] and benzodiazepines [6]. conditions [12], mainly at the frozen temperatures. Statistical anal- 50

35 Stability study in biofluids is an important prerequisite for ysis was applied to evaluate the data uncertainty using linear or 51

36 validation of a bioanalytical method [7] and has practical implica- non-linear methods [13]. 52

37 tions in the drug determination during pharmacokinetic evaluation Application of the short-term stability studies can be an alterna- 53

38 [8] and can avoid error in bioanalytical laboratory in the con- tive to reduce the cost of the bioanalytical validation program and 54

39 text to assurance a more effective patient management, including to evaluate the behavior of drugs in biological matrices. This article 55

presents some analytical data of beta-lactams in stock solution and 56

human plasma at different temperatures during short-term stabil- 57

ity studies. The purpose of this study was to apply the Arrhenius 58
∗ Corresponding author. Tel.: +55 021 82 3214 1155; fax: +55 021 82 3214 1155.
equation in the stability study of ampicillin and cephalexin in order 59
E-mail addresses: ticianogn@yahoo.com.br (T.G. do Nascimento),
to determine adequate temperature and time of storage of these 60
eoliveira@gmail.com (E. de Jesus Oliveira), ruiomacedo@yahoo.com.br
(R.O. Macêdo).
drugs using an appropriate statistical analysis. 61

0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2012.04.010

Please cite this article in press as: T.G. do Nascimento, et al., Short-term stability studies of ampicillin and cephalexin in aque-
ous solution and human plasma: Application of least squares method in Arrhenius equation, J. Pharm. Biomed. Anal. (2012),
http://dx.doi.org/10.1016/j.jpba.2012.04.010
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2 T.G. do Nascimento et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2012) xxx–xxx

62 2. Experimental CQ2 and CQ3 , were transferred into cryovials and stored at 20 ◦ C, 119

2 ◦ C and −20 ◦ C during 15 days, 30 days and 103 days, respectively, 120

63 2.1. Reagents and chemicals to promote sample aging. Non-spiked fresh plasma samples were 121

also stored in the same conditions and at the moment of analysis 122

64 Ampicillin USP (from Ferjinsá, Mexico) and cephalexin phar- were spiked with analytes in order to obtain in all the samples the 123

65 macopeial standard (from Brazilian Pharmacopeia) were used same nominal concentration of the aged samples and calibration 124

66 throughout the study. Perchloric acid, phosphoric acid, potas- curve samples. Stability of beta-lactams was evaluated by compar- 125

67 sium dihydrogen phosphate and HPLC-grade acetonitrile were ing the responses of ampicillin and cephalexin in stored samples 126

68 purchased from Merck, Darmstadt, Germany. Water was purified and with those freshly prepared sample at the same nominal con- 127

69 with a reverse osmosis system connected to an ion exchange unit centration level. An upper and lower limit of 10% of the original 128

70 (Gehaka, Brazil). All other reagents were analytical grade. concentration was established as acceptance criteria for stability. 129

All analyses were done in triplicate. 130

71 2.2. LC-DAD and LC-UV–MS/MS conditions

2.5. Application of Arrhenius equation and statistical analysis 131

72 The HPLC system consisted of an LC-10AD pump, a column

73 oven (model CTO-10AS), a diode array detector (SPDM10A), an Each quality control sample (QC1 , QC2 and QC3 ) was used to 132

74 autosampler SIL-10AD, and a controller module SCL-10A (all from calculate the order of reaction and degradation rate constant (k) in 133

75 Shimadzu) coupled to a personal computer running the software each temperature studied 20 ◦ C, 2 ◦ C and −20 ◦ C using Arrhenius 134

76 Shimadzu Class VP for data acquisition. The mobile phase consisted equation as time function (Eqs. (1)–(3)) [15]. In each temperature, 135

77 of acetonitrile: dihydrogen phosphate (pH 3.5; 0.1 M) (12.5: 87.5, the mean of the degradation rate constant (k) (from triplicate exper- 136

78 v/v). The pH of the phosphate buffer was adjusted with 4% phos- iments) was obtained and did not presented variation major than 137

79 phoric acid. Separation was achieved at 25 ◦ C using a Shimpak C18 15% of relative standard deviation (RSD). 138

80 column (250 mm × 4.6 mm; i.d. 5 ␮m) fitted with a Phenomenex C0 − Cx

81 security guard C18 column (4.0 mm × 3.0 mm; i.d. 5 ␮m). The flow k0 = (1) 139
t x − t0
82 rate was set at 1.0 mL min−1 and a discrete channel on the diode
83 array detector configured to acquire data at 215 nm. Samples were Cx /C0
k1 = ln (2) 140
84 introduced using the autosampler and the injection volume was tx − t0
85 40 ␮L. (1/Cx ) − (1/C0 )
86 The LC-UV–MS/MS conditions for detection of the beta-lactams k2 = (3) 141
tx − t0
87 included a mobile phase that consisted of acetonitrile: formic acid
(0.1%) (10:90, v/v) delivered at a flow rate of 1.0 mL min−1 that was A − Ea
88 ln k = ln (4) 142
89 reduced with a splitter to 0.2 mL min−1 for introduction into the RT
90 mass spectrometer. Separation was achieved using a Shimpak C18 where C0 = initial concentration, Cx = concentration of the drug in 143

91 Column (250 mm × 4.6 mm; i.d 5 ␮m) fitted with Shimpak secu- final time, t0 = initial time; tx = final time, k0 = zero-order degra- 144

92 rity guard C18 column (4.0 mm × 3.0 mm; i.d. 5 ␮m) kept at 40 ◦ C dation rate constant, k1 = first-order degradation rate constant, 145

93 in a column oven. A triple quadrupole mass spectrometer (Quat- k2 = second-order degradation rate constant, A = preexponential 146

94 tro LC, Micromass) was used for determination of ampicillin, its factor, Ea = energy of activation, R = gas constant, T = absolute tem- 147

95 related degradation products and cephalexin. It was operated in perature in Kelvin. 148

96 positive electrospray ionization mode with a capillary voltage of The degradation rate constant (k) at sub-zero temperature was 149

97 2.70 kV, sampling cone voltage of 21 V, extraction cone voltage of estimate by extrapolation of the best linear regression with least 150

98 3 V, desolvation gas temperature of 250 ◦ C (N2 ) and source block squares method for the temperature of −30 ◦ C and −40 ◦ C, which 151

99 temperature of 110 ◦ C. Full scan acquisition was made in the range were obtained according to graphical analysis using Arrhenius 152

100 of 40–400 m/z with the multiplier voltage at 650 V. equation (4) and the time of storage was also calculated [15]. It was 153

calculated the coefficient of correlation (r2 ), residual analysis and 154

101 2.3. Sample preparation estimating the standard error (SYX ) in the Arrhenius plot (ln k × 1/T) 155

using the least squares method. The 95% confidence interval and 156

102 2.3.1. Human plasma samples 95% prediction interval were used to obtain the upper and lower 157

103 Human plasma (250 mL) collected from healthy volunteers and limits of variations [16]. Analysis of variance (ANOVA-one way) and 158

104 stored in bags containing heparin as anticoagulant was acquired P-value were used to check if the experimental values followed the 159

105 from the Hemotherapy Center of Paraíba (João Pessoa, Paraíba, Arrhenius plot. 160

106 Brazil) after approval from the research ethics committee. The
107 method for quantification of ampicillin and cephalexin during the 3. Results and discussion 161

108 stability studies has been previously validated [14].

3.1. Stability studies in stock solution 162

109 2.3.2. Stock solution

110 Ampicillin and cephalexin (50 mg) were weighed and quantita- During the stability study in stock solution the appearance 163

111 tively transferred to a 10 mL volumetric flask to obtain a (5 mg/mL) of two new chromatographic peaks at 5.5 and 6.1 min became 164

112 stock solution in ultrapure water. Successive dilutions were made apparent in the period after 15 days of storage. These peaks were 165

113 to prepare quality control samples at concentrations of 1.0, 2.5, 5.0, denominated AMP A1 and AMP A2. Additional experiments were 166

114 10.0 and 50.0 ␮g/mL. The stock solutions were stored in cryovials done to determine the origin of the degradation products. Two 167

115 of 2.0 mL. stock solutions containing only one analyte were submitted to 168

aging for 10 days at 20 ◦ C and analyzed. Degradation products 169

116 2.4. Stability studies in stock solution and human plasma appeared in the ampicillin stock solution (10 ␮g/mL) but not in 170

cephalexin stock solution (10 ␮g/mL). Thus, degradation products 171

117 Aliquots of 1.0 mL from the quality control samples (5.0, 10.0 (AMP A1 + AMP A2) were formed uniquely from ampicillin after 172

118 and 50.0 ␮g/mL) in stock solution or human plasma, named CQ1 , 10 days of storage at 20 ◦ C (Figs. 1 and 3A and B). 173

Please cite this article in press as: T.G. do Nascimento, et al., Short-term stability studies of ampicillin and cephalexin in aque-
ous solution and human plasma: Application of least squares method in Arrhenius equation, J. Pharm. Biomed. Anal. (2012),
http://dx.doi.org/10.1016/j.jpba.2012.04.010
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T.G. do Nascimento et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2012) xxx–xxx 3

Fig. 2. 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. Chromatographic conditions acetonitrile:formic acid
(0.1%) (10:90, v/v). Chromatograms in MS/MS detection (A) and (B) in scan mode
(50–400 m/z) and chromatogram in UV detection (C).

(AMP A2) in the extracted ion chromatograms and the mass spec- 194

tral presented this evidence and has confirmed the formation of 195

two epimeric forms, 1 (5S, 6R) and 2 (5R, 6R), of the ampicilloic acid 196

from the ampicillin degradation. The conversion of the ampicillin in 197

ampicilloic acid 1 (5S, 6R) and 2 (5R, 6R) in acid aqueous medium 198

can be demonstrated during sample preparation of ␤-lactams in 199

stock solution. The opening of the ␤-lactamic ring by acid hydrolysis 200

is the accepted mechanism for formation of these two ampicil- 201

loic acid diastereoisomers (Fig. 3). Zhu et al. [17] reported several 202

degradation products from ampicillin including epimeric forms of 203

ampicilloic acid, in which the ampicilloic acid (5S, 6R) exhibited 204

a lower retention time than its (5R, 6R) diastereoisomer and our 205

data have corroborated these results [18]. It is reported degradation 206

of cephalexin to 7-aminodesacetoxicephalosporanic acid usually 207

found when the drug is subjected to extreme conditions such as 208

Fig. 1. (A) Chromatogram of drug-free plasma (a) and spiked plasma (b) with (1) acidic or alkaline conditions (pH 1 or pH 8) and in this conditions 209
ampicillin (10 ␮g/mL) and (2) cephalexin (10 ␮g/mL). (B) Chromatogram of stock cephalexin is 180 times more stable than ampicillin [19]. 210
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). (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.

174 LC-UV–MS/MS experiments were done in order to identify the

175 degradation products (AMP A1 and AMP A2) (Fig. 2). Stock solution
176 containing ampicillin and cephalexin aged during 15 days at 20 ◦ C
177 were analyzed using full scan mode. The LC-UV chromatogram
178 showed presence of AMP A1, AMP A2, ampicillin and cephalexin
179 at retention times of 8.0, 9.0, 14.5 and 15.4 min, respectively, in
180 agreement with a previously validated method [14]. The extracted
181 ion chromatograms displayed two peaks at m/z 368 with retention
182 times of 8.0 and 9.0 min and a peak with m/z 350 at the retention
183 time corresponding to ampicillin.
184 The scan mode (using collision-induced dissociation) allowed
185 obtaining more information about the fragmentation pattern of
186 the ions of ampicillin (m/z 350) and theirs degradation products
187 (m/z 368). The mass spectra of both AMP A1 and AMP A2 presented
188 a mass gain of 18 amu in relation to ampicillin, which suggests a
189 hydrolytic reaction involving ampicillin and a water molecule. The
190 spectra also presented an uncommon ion (at m/z 324) that is related
191 to the loss of carbon dioxide (CO2 ) and suggests the presence of
192 a second carboxylic acid group in the molecule. The detection of Fig. 3. Scheme illustrating the ampicillin conversion in two diastereoisomeric forms
193 ions at m/z 368 with retention times 8.0 (AMP A1) and 9.0 min of the ampicilloic acid, AMP A1 (5S, 6R) and AMP A2 (5R, 6R) in aqueous medium.

Please cite this article in press as: T.G. do Nascimento, et al., Short-term stability studies of ampicillin and cephalexin in aque-
ous solution and human plasma: Application of least squares method in Arrhenius equation, J. Pharm. Biomed. Anal. (2012),
http://dx.doi.org/10.1016/j.jpba.2012.04.010
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Fig. 4. Quantitative loss mass of ampicillin (A) with simultaneous conversion in two degradation products AMP A1 and AMP A2 in stock solution; (B) quantitative loss mass
of cephalexin in stock solution. Stability studies of ampicillin and cephalexin in human plasma (C) storage at room temperature, (D) storage at 2 ◦ C, (E) storage at −20 ◦ C and
(F) after freeze–thaw cycles. 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.

211 3.2. Stability studies in human plasma which was observed in the fourth cycle of freezing and thawing 230

(Fig. 4F). 231

212 The stability study of ampicillin and cephalexin spiked in human

213 plasma revealed degradation of the drugs after 6 h of storage at 3.3. Application of Arrhenius equation and statistical analysis 232
214 20 ◦ C. At 24 h of storage ampicillin and cephalexin presented degra-
215 dation greater than 10% (Fig. 4C). Ampicillin and cephalexin spiked The data showed a similar kinetic of degradation at 20 ◦ C 233
216 in human plasma presented degradation levels close to 40% after (second-order kinetic) and at low temperatures, 2 ◦ C and −20 ◦ C, 234
217 15 days in the stability study at 2 ◦ C (Fig. 4D). At freezer tempera- a first-order kinetic for both beta-lactams. The degradation rate 235
218 ture (−20 ◦ C) the degradation of ampicillin is delayed for 10 days, constant of the ampicillin and cephalexin were assessed as time 236
219 whereas cephalexin was stable for 20 days within limits established function (Fig. 4C–E), Arrhenius plot was applied to assure a more 237
220 (<10%) (Fig. 4E). During the stability study, the formation of interfer- long time of storage for beta-lactamics in human plasma and to 238
221 ents from the plasma aging process (30 days at room temperature; avoid degradation of drugs during the analysis. The ideal time of 239
222 60 days at 2 ◦ C; and 103 days at −20 ◦ C) resulted in coelution with storage involving processing and analysis of extracted samples is 240
223 cephalexin. Thus, conditions for cephalexin quantification are more shown in Table 1. 241
224 restrictive than ampicillin. It was calculated the extrapolated degradation rate constants 242
225 The study of freezing and thawing cycles showed the ampicillin (k1 ) values at sub-zero temperature and the maximum time of 243
226 and cephalexin were stable for three consecutive cycles within storage to the analytes, ampicillin and cephalexin, using the (k1 ) 244
227 limits (<10%). The study of freeze–thaw cycles also revealed a first order degradation constant (Table 1). The k1 values were 245
228 high variability of the measurements (>15%) after the third cycle extrapoled applying the least squares method into the Arrhe- 246
229 and lower recovery of ampicillin (84.7%) and cephalexin (88.9%) nius plot ln k × (1/T) with the best fit observed by coefficient of 247

Please cite this article in press as: T.G. do Nascimento, et al., Short-term stability studies of ampicillin and cephalexin in aque-
ous solution and human plasma: Application of least squares method in Arrhenius equation, J. Pharm. Biomed. Anal. (2012),
http://dx.doi.org/10.1016/j.jpba.2012.04.010
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Table 1
Q5 Determination of order of reaction, degradation rate constants and estimative of storage time at sub-zero temperature for ampicillin and cephalexin applying the least
Q4
squares method in Arrhenius equation.

Temp. (◦ C) Zero order First order Second order

k0 r2 k1 r2 k2 r2

Ampicillin
20 8.77 ± 0.87 0.983 0.1157 ± 0.015 0.990 0.00157 ± 0.00027 0.995
2 2.88 ± 0.50 0.966 0.0381 ± 0.0090 0.962 0.000523 ± 0.0016 0.953
−20 0.81 ± 0.18 0.991 0.0097 ± 0.0013 0.987 0.000109 ± 0.000032 0.982

Cephalexin
20 8.79 ± 1.39 0.989 0.1237 ± 0.0024 0.993 0.00171 ± 0.00005 0.993
2 2.32 ± 0.14 0.987 0.0312 ± 0.0025 0.974 0.000432 ± 0.00005 0.956
−20 0.42 ± 0.12 0.910 0.0054 ± 0.0002 0.899 0.000061 ± 0.000006 0.878

Temp. (◦ C) Ampicillin Cephalexin

k1 Time (days) k1 Time (days)

20 0.00157a 0.64 0.00171a 0.58

2 0.0381b 2.7 0.0312b 3.4
−20 0.0097b 10.8 0.0054b 19.4

Statistical parameters

Ampicillin Cephalexin

Linear regression y = 13.387 − 4568.1x ± 213 Linear regression y = 17.609 − 5781.3x ± 177
r2 0.9978 r2 0.9991
SYX 0.08169 SYX 0.06788
ln k (−40 ◦ C) −6.219 ± 0.1412 ln k (−40 ◦ C) −7.204 ± 0.1173
95%CI of ln k(−40 ◦ C) −8.013 to −4.425 95%CI of ln k(−40 ◦ C) −8.695 to −5.713
F 469.7 F 1066
P < 0.0500 0.0297 P < 0.0500 0.0195

Prediction of Arrhenius parameters

Ampicillin Cephalexin

Temp. (◦ C) k1 b Time (days) Temp. (◦ C) k1 b Time (days)

−30 0.004468 23.5 −30 0.002068 50.8

−40 0.001991 52.7 −40 0.000744 141.2

Each value is expressed as mean ± SD of three determinations of the QC samples. k0 = (C0 − Cx )/(tx − t0 ); k1 = ln[Cx /C0 ]/(tx − t0 ); k2 = [(1/Cx ) − (1/C0 )]/(tx − t0 ).
a
k2 = second-order degradation rate constant; t2(90) = 0.001/k2 .
b
k1 = first-order degradation rate constant; t1(90) = 0.105/k1 .

248 correlation and residual analysis, which was calculated to deter- has reduced the hyperbolic amplitude on the regression analysis 275

249 mine the experimental error of the best linear curve for ampicillin [16]. 276

250 (6.6%) and cephalexin (5.5%). In some cases, linear methods have Ampicillin and cephalexin could be preserved for a more long 277

251 some advantages over the non-linear methods because they are time of storage at sub-zero temperature (−30 ◦ C and −40 ◦ C), what 278

252 robust to deviations from homoscedasticity [13]. Fig. 4G and H would be sufficiently to preserved ampicillin for periods of between 279

253 showed the best regression curve with application of the estimative 20 and 50 days, while cephalexin would be preserved for 52 and 280

254 of confidence interval for the extrapolation calculi of the (k1 ) val- 141 days, respectively. The extrapoled degradation rate constants 281

255 ues of ampicillin and cephalexin at sub-zero temperature (−40 ◦ C). (k1 ) values of ampicillin and cephalexin presented relative standard 282

256 Estimative of confidence interval of degradation rate constant (k1 ) deviation (RSD) of 2.3% and 1.6%, respectively, as demonstrated in 283

257 was extrapolated for temperatures of −50 ◦ C, but there was no suc- Table 1 and thus adequate in the previsibility of the extrapolated 284

258 cess in the calculi [16]. The analysis of variance (F) and P value time of storage. 285

259 demonstrated significant difference in relation to the values (k1 ),

260 thus the degradation rate constants values obeys the Arrhenius law 4. Conclusion 286

261 and were dependent on the storage temperature [15] (Table 1).
262 The best regression curve presented in Fig. 4G and H has demon- Ampicillin and cephalexin were presented instability in human 287

263 strated good estimative of standard error (SYX ) for ampicillin (8.2%) plasma after short periods of storage at temperature of −20 ◦ C. 288

264 and cephalexin (6.8%). Ampicillin presented greater variation in Based on this special class of drugs very unstable in biological flu- 289

265 (SYX ) and thus resulting in major amplitude in the hyperbolic effect ids it was considered the propose to determinate the adequate time 290

266 of the confidence bands in relation to cephalexin. The planning and temperature of storage in short-term stability studies during 291

267 of this stability studies only in three different temperatures was the step of method validation. 292

268 not sufficient to minimize the amplitude of the hyperbolic effect of The beta-lactams, ampicillin and cephalexin, when submitted to 293

269 confidence bands. The minimum sample size of (k1 ) and the esti- natural processes of degradation in aqueous medium or biological 294

270 mative of standard error (SYX ) contributed for the large confidence conditions has attendant to the classical Arrhenius method. The 295

271 interval of the ln k (−40 ◦ C) for ampicillin (28.8%) and cephalexin Arrhenius equation was useful to evaluate the degradation kinetic 296

272 (20.7%) (Table 1). Substitution of the mean values of rate constant of beta-lactams and estimate the ideal time and temperature of 297

273 (k1 ) in each temperature by individual values of rate constants storage, but statistical tools must be employed to avoid incorrect 298

274 (k1 ) obtained for each quality control samples (CQ1, CQ2 and CQ3) extrapolations and estimated values out uncertainty limits. 299

Please cite this article in press as: T.G. do Nascimento, et al., Short-term stability studies of ampicillin and cephalexin in aque-
ous solution and human plasma: Application of least squares method in Arrhenius equation, J. Pharm. Biomed. Anal. (2012),
http://dx.doi.org/10.1016/j.jpba.2012.04.010
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300 Acknowledgements M. Laurentie, J.C. Nivet, The SFSTP guide on the validation of chromatographic 328
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Please cite this article in press as: T.G. do Nascimento, et al., Short-term stability studies of ampicillin and cephalexin in aque-
ous solution and human plasma: Application of least squares method in Arrhenius equation, J. Pharm. Biomed. Anal. (2012),
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