Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development of a stability-indicating HPLC method of Etifoxine with characterization of degradation products by LC-MS/TOF, 1 H and 13 C NMR Nadia Djabrouhou, Moulay-Hassane Guermouche ∗ Laboratoire de chromatographie, Faculté de chimie, USTHB, BP 32, El-Alia, Bab-Ezzouar, 16133 Alger, Algeria
a r t i c l e
i n f o
Article history: Received 10 May 2014 Received in revised form 18 July 2014 Accepted 20 July 2014 Available online 27 July 2014 Keywords: HPLC Etifoxine Stress degradation LC-MS/TOF 1 H and 13 C NMR Validation
a b s t r a c t This paper describes a new LC-MS/TOF method for the degradation products determination when Etifoxine (ETI) is submitted to different stress conditions. Chromatography is performed by using Kromasil C18 column (250 mm × 4.6 mm, 5 m particle size). The selected mobile phase consists of formate buffer 0.02 M, pH 3 and methanol (70/30, v/v). ETI is submitted to oxidative, acidic, basic, hydrolytic, thermal and UV light degradations. Detection is made at 254 nm by photodiode array detector and mass spectrometry. A number of degradation products (DPs) called DPA, DPB, DPC and DPD are found depending on the stress; DPA with heat, DPA and DPB in acidic media or under UV-light; DPA, DPB and DPC under basic stress; DPA, DPB, DPC and DPD with oxidation. LC-MS/TOF is used to characterize the four DPs of ETI resulting from different stress conditions. 1 H and 13 C NMR are used to confirm the DP structures. The ETI fragmentation pathway is proposed. The method is validated with reference to International Conference on Harmonization guidelines and ETI are selectively determined in presence of its DPs, demonstrating its stability-indicating nature. Finally, for the validation step, specificity, linearity, accuracy and precision are determined for ETI and its DPs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Etifoxine (ETI), is chemically designated as 6-chloro-N-ethyl4-methyl-4-phenyl-4H-benzo[d][1,3]oxazin-2-amine. It is used as an anxiolytic and anticonvulsant drug [1]. It has similar effects to benzodiazepine drugs with different side effects [2,3]. Differential effects of Etifoxine on anxiety-like behavior and convulsions in mice have been recently described in literature [4]. In another work, the authors demonstrate the role of 5-HT2 receptor subtypes in the mechanism of action of the GABAergic compound Etifoxine in mice [5]. The anxiolytic ETI protects against convulsant and anxiogenic aspects of the alcohol withdrawal syndrome are also studied [6]. Some authors investigate the impact of ETI on lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety [7]. A number of studies have been conducted and reported in recent years on the biological and pharmacological action of ETI. However, to the best of our knowledge, very few research papers tackle the problem of quantitative analysis of ETI. The unique relevant paper (in Chinese) on ETI analysis in
∗ Corresponding author. Tel.: +213 21247311; fax: +213 21247311. E-mail address: [email protected] (M.-H. Guermouche). http://dx.doi.org/10.1016/j.jpba.2014.07.017 0731-7085/© 2014 Elsevier B.V. All rights reserved.
pharmaceuticals that we identified presents a non-validated method which combines an Agilent TC-C18 column with mobile phase constituted with methanol–water (70:30, v/v) [8]. Another general work on screening procedure for the characterization of human drug metabolites briefly cited the case of Etifoxine [9]. Therefore, it is imperative to develop a rapid and simple procedure of ETI HPLC analysis. In recent years, it is strongly recommended that any HPLC development method of active pharmaceutical ingredients should include a good peak separation between a drug and its DPs; official methodology exists [10]. A great number of papers describe this type of studies for several drugs using HPLC or LC-MS-MS. The stress degradation are performed on Clobetasol 17-propionate [11], Esomeprazole [12], Entecavir [13], Abiraterone [14], Indinavir sulphate [15], Ranitidine [16], Desipramine hydrochloride [17], Cefpirome [18] and Dimethindene Maleate [19]. More recently, stress degradations of Metaxalone [20], cephalothin [21] and Mometsone Furoate [22] are studied. Moreover, recent study proposes a critical review of the HPLC-MS use in this field [23]. To the best of our knowledge, there is no stability-indicating method of ETI. The ultimate objective of the present work is to present a new and validated stability-indicating HPLC method for the quantitative determination of ETI in capsules involving an
12
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
acidic, basic, oxidative, thermal and light degradation of ETI. The chemical structures of the different DPs produced by stress are investigated by LC-MS/TOF and 1 H and 13 C NMR.
tailing factor, resolution between ETI and DPs) are measured for each mobile phase. 2.4. Stress studies
2. Experimental 2.1. Reagents Etifoxine hydrochloride (99.3%) is kindly provided by the National Laboratory Control of Pharmaceutical Products (Algiers, Algeria). The substance is used as received for preparing standard solutions. Ammonium formate is obtained from Prolabo (France), formic acid and LC quality methanol are purchased from Sigma Aldrich (USA). 2.2. Instrumentation For the LC-MS/TOF experiments, Ultimate 3000 chromatograph (Dionex, USA) coupled to MicroTof Q II (Brüker Daltonics, Germany) is used. Chromatography is performed on the same conditions selected in LC experimentation. The following LC-MS/TOF parameters are used: source type, ESI; ion polarity, positive; end plate offset voltage, −500 V; capillary voltage, 4000 V; nebulizer pressure, 69.6 psi, dry gas, 12.0 L min−1 ; dry heater, 300 ◦ C; collision cell RF, 150.0 Vpp; scan begin 50 m/z; scan end, 600 m/z. Data are collected on Compass (Brüker Daltonics, Germany) software. LC analysis is performed with a Waters chromatograph equipped with a 600E pump, 7725i Rheodyne injector with 20 L sample loop, photodiode array detector. ETI is eluted on a KROMASIL C18 analytical column (250 mm × 4.6 mm i.d., 5 m particles) from AKZO Nobel (Sweden) under reversed phase partition chromatographic conditions. The flow rate is 1.0 mL min−1 and the analyte is monitored at the maximal absorption of ETI (254 nm). Data are collected with Empower program (Waters). Before the analysis, the mobile phase is degassed by using a Branson sonicator (Branson Ultrasonics, USA) and filtered through a 0.45 m filter. The system is equilibrated before each injection. NMR experiments are performed using a 400-MHz Brüker NMR Spectrometer, with TMS as a reference and CDCl3 as solvent of ETI and DPs samples. 2.3. Determination of the optimal HPLC conditions Several trials are carried out to obtain a good resolution between the drug and its DPs. These trials involved the use of different mobile phases with different ratios, and different pH. The suitable mobile phase should control the selectivity and achieve reproducible separations with an acceptable peak shape. It is prepared daily and filtered through a 0.45 m Millipore filter and ultrasonicated for 30 min before use. Ammonium acetate has a pKa of 4.8 and its buffer range (pKa ±1 unit) is 3.8 and 5.8. Therefore, the mobile phases are made of methanol (volumetric composition varied from 65 to 75%) and formate buffer 0.02 M (pH from 3 to 4). The use of methanol was important to increase the retention factor and to obtain a better resolution of drug and DPs. Four mobile phases are tested: Mobile phase 1: formate buffer 0.02 M (pH 4)–MeOH, 35–65, v/v. Mobile phase 2: formate buffer 0.02 M (pH 3)–MeOH, 30–70, v/v. Mobile phase 3: formate buffer 0.02 M (pH 3)–MeOH, 25–75, v/v. Mobile phase 4: formate buffer 0.02 M (pH 3.5)–MeOH, 25–75, v/v. Flow rate is fixed to 1 mL min−1 . Injections are made in triplicate. Chromatographic parameters (retention time, plate number and
The stability of ETI under the conditions of hydrolysis, oxidation and thermal degradation are investigated as mentioned in International Conference on harmonization [24]. In each case, the stress time is maintained long enough to achieve at least 20% degradation of the drug initial amount. The stress experiments are made with an initial solution containing ETI at 2 mg mL−1 of ETI in methanol except for thermal and photolytic degradation. Acidic degradation is carried out by adding 1 mL of 2 M HCl to 1 mL of the initial condition; 20% of ETI is degraded after 60 h. Alkali degradation is performed by mixing 1 mL of initial solution and 1 mL of 2 M NaOH; 120 h are necessary to obtain 20% of degadation. 20% of oxidative degradation is obtained by exposing 1 mL of initial solution to 1 mL of 3% (v/v) H2 O2 during 60 h. Twenty percent of ETI degradation with heat was performed by heating pure ETI at 80 ◦ C during 4 h. Light degradation is obtained by submitting ETI to a light from Mercury lamp (50 W Mercury high pressure lamp, luminous flux 2000 Im) during 24 h. Before LC/MS, solutions resulting from stress are diluted 50 times with mobile phase, filtered under 25 mm Millex-HV membrane syringe filter (pore size 0.25 m). The samples of ETI degraded under different conditions are isolated by semi-preparative HPLC using Kromasil C18 (250 mm × 10 mm, 5 m) column with the same mobile phase selected after the determination of the optimal chromatographic conditions. Injection volume of the sample is fixed to 200 L. The column eluents are monitored by a photo diode array detector at 254 nm. Four collected solutions containing DPA, DPB, DPC and DPD respectively are extracted with 20 mL of chloroform, evaporated to dryness. The residue of each solution with one DP is collected and used for the validation assays and NMR experiments. 2.5. Validation of the HPLC method Specificity of the developed LC method for ETI is examined in the presence of its degradation products (namely DPA, DPB, DPC and DPD) and the excipients present in capsules (lactose monohydrate, talc, microcrystalline cellulose, colloidal anhydrous silica and magnesium stearate). A placebo capsule matrix solution is prepared from a weighted mixture of the excipients without the drug respecting their weight composition in the commercial capsules. It is mixed with oxidative stress sample solution to improve specificity because hydrogen peroxide action on ETI generates four DPs. The limit of detection (LOD) and quantification (LOQ) for ETI and its four DPs are determined at a signal-to-noise ratio of 3 and 10, respectively. Linearity is carried out with external standard method for ETI and its DPs. Five standard solutions of ETI (10, 15, 20, 25 and 30 g mL−1 ) are prepared separately in methanol. These solutions are made daily and analyzed immediately after preparation. They correspond to 50%, 75%, 100%, 125% and 150% of ETI. For each DP, the linearity is examined from 0.5 to 4 g mL−1 (4 g mL−1 corresponds to 20% of ETI degradation). All solutions are filtered using a 25 mm Millex-HV membrane syringe filter (pore size 0.45 m). Twenty microliter of each solution is injected in triplicates. The peak area of ETI and DPs are measured, a calibration curves are obtained from the least-squares linear regression of the identified peak area. Precision is evaluated as the repeatability and intermediate precision levels. Six samples (100% level: 20 g mL−1 for ETI and 4 g mL−1 for each DP) are freshly prepared and triplicates
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
13
Fig. 1. (a) Bar graphs showing the variation of: (a) ETI chromatographic parameters with methanol composition and pH; (b) resolution between DPs and ETI with methanol composition and pH.
measurements are used to perform repeatability. Intermediate precision is calculated from data of six independent standards with a level of 100% (20 g mL−1 for ETI and 4 g mL−1 for each DP). These standards are analyzed by two independent analysts, in triplicates, during three different days. Accuracy is measured at concentration levels of 50% (three samples), 100% (three samples) and 150% (three samples) where a known amount of the active is added to an amount of placebo. Accuracy is estimated with ETI or degradation product RSD%.
3. Results and discussion 3.1. Determination of the optimal chromatographic conditions All the results are given in Fig. 1a. To select the optimal chromatographic conditions, plate number effect is examined first, tailing factor of ETI peak is the second parameter considered. The mobile phase made of formate buffer 0.02 M (pH 3)–MeOH, 30–70, v/v gives the greatest plate number (N = 5081), the lowest tailing factor value (1.06). The corresponding retention time is convenient (retention time = 7.5 min). On the other hand, using the different tested mobile phases, resolutions between ETI and the DPs given by the stressed sample solution resulting from oxidative stress are calculated and plotted in Fig. 1b. With all the mobile phases, elution order is DPC, ETI and DPD, DPB and finally DPA. The best resolution between DPC and ETI is given by the mobile phase 1 but the DPA retention time exceeds 43 min. When we consider the resolutions between ETI and DPD, DPD and DPB, DPB and DPA, the greatest values are given by the mobile phase 2. Moreover, with this solvent, the A retention time is convenient and around 20 min. Finally, we select the mobile phase 1 made of 70% of methanol in formate buffer 0.02 M (v/v) adjusted to pH 3. Suitability parameters found including plate number, tailing factor and retention factor, comply with the FDA guidelines on Validation of Chromatographic Methods [25].
3.2. Forced degradation studies 3.2.1. Stress degradation by hydrolysis under acidic conditions In the conditions described in Section 2, 20% degradation occurs after about 70 h. The corresponding LC chromatogram is given in
Fig. 2a. Two DPs called DPA and DPB are observed at 21 and 13 min respectively. 3.2.2. Stress degradation by hydrolysis under basic conditions It seems that ETI degradation is slower in basic medium compared to acidic solution. In basic solution, with the conditions described in the experimental part, 20% of ETI degradation is not reached after 120 h of stress. Fig. 2b illustrates the corresponding chromatogram. We observe three DPs; two of them have the same retention times of DPA and DPB found under the acidic attack. The third DP called DPC has 4 min as retention time. 3.2.3. Oxidative stress ETI is found to be more sensitive to stress with 3% H2 O2 than to acidic or basic media. About 45 h of exposure are required to obtain 20% degradation with 3% H2 O2 . Fig. 2c shows the corresponding chromatogram where four DPs are found. Three of them are already detected at the same times when ETI is submitted to acidic (DPA and DPB) or basic stress (DPA, DPB and DPC). The new degradation product called DPD has a 10 min retention time. 3.2.4. Thermal degradation The highest stress effect is found with the temperature action. After 2 h of heat at 80 ◦ C, 20% of ETI are degraded leading to the formation of only one DP having the same retention time of DPA. Chromatogram appearing in Fig. 2d is relative to this effect. 3.2.5. Degradation under UV light Compared to the other stress parameters, UV light effect on ETI is faster than acidic stress; it produces two DPs at the same retention time than DPA and DPB (Fig. 2e) and 20% of ETI are lost after 24 h. Fig. 2f shows UV spectra of DPA, DPB, DPC and DPD recorded by PDA detector. The DPs found with the other stress give the same spectra (DPA and DPB in acidic media and after photolytic action; DPA, DPB and DPC with basic stress; DPA after thermal degradation). As the DPs have the same retention times and the same UV spectra with all stresses, we can assume that the DP structures can be the same.
14
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Fig. 2. Chromatograms ETI stress degradation with HCl (a), NaOH (b), H2 O2 (c), heat (d) and photolytic action (e). UV spectra of ETI and the DPs A, B, C and D (f).
3.3. LC-MS/TOF, 1 H and 13 C NMR results LC-MS/TOF is used to identify the DPs resulted from ETI degradation. It is important to remember that under H2 O2 stress, ETI gives four degradation products DPA, DPB, DPC and DPD at approximately 21, 13, 4 and 10 min as retention times; ETI is eluted in about 7 min. Mass spectra of ETI, DPA, DPB, DPC and DPD are given in Fig. 3 in the rectangles reported as ETI, Degradation product A, Degradation product B, Degradation product C and Degradation product D respectively. In basic media, three DPs appear. They have the same retention times, UV spectra and mass spectra than the DPA, DPB and DPC found with oxidative stress. We can conclude that it is most likely the same products. The same reasoning can be held with
other stress; thermal attack gives DPA only while DPA and DPB are obtained with acidic or UV stress. The unique other DP is done by oxidative action. These conclusions must be confirmed by LCMS/TOF, 1 H and 13 C NMR. For ETI, DPA DPB DPC and DPD, mass spectra are given if Fig. 3 where hydrogens and carbons are identified by numbers and letters respectively. In Fig. 4, 1 H and 13 C NMR (including J-mode) spectra of ETI, DPA DPB DPC and DPD are presented. Their corresponding chemical shifts are given in Table 1. ETI mass spectrum of DPA is given in Fig. 3. According to mass spectrum formula report, the ion with 301 molecular weight corresponds to a proposed formula C17 H18 ClN2 O equivalent protonated molecular ions [M + H]+ at m/z = 301.1121. ETI MS/TOF spectrum of [M + H]+ ions (m/z 301) eluting at approximately 7 min retention
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
15
Fig. 3. Mass spectra of ETI (ETI, rectangle), DPA (A, rectangle), DPB (B, rectangle), DPC (C, rectangle) and DPD (D, rectangle). Numbers and letters assigned to H and C atoms present in ETI, DPA, DPB, DPC and DPD.
time displays product ions at m/z 256.0481 and 230.0749 Da; their best proposed formula are C15 H11 NOCl+ and C14 H13 NCl+ respectively. Their proposed structures are given in Table 1. The 1 H NMR spectra of ETI and the corresponding proton chemical shifts are presented in Fig. 4 and Table 2 respectively. Mass spectrum of degradation product A (retention time about 21 min) appears with all stress with a pseudomolecular ion peak [M + H]+ at m/z 301.1096. It is surprising to note that ETI and DPA have the same molecular mass. Comparing the mass spectra of
ETI and DPA, It is important to note that the ETI fragment located at 256.0481 does not exist in DPA which suggests that probably, the oxazin-2-amine cycle does not exist in DPA, it undergoes an opening the oxazine ring present in ETI giving DPA with formation of carbonyl and ethylenic functions. DPA chemical name is 1-(5-chloro-2-(3-ethylureido) phenyl)-1-phenylethylene. Another fragment found (m/z = 230) is common to ETI and DPA. Its structure is given in Fig. 3 and must be confirmed by NMR. In Fig. 4, we present 1 H NMR and 13 C spectra of DPA and its corresponding
16
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Fig. 4.
1
H and 13 C NMR (including J-mode spectra) of ETI, DPA, DPPB, DPC and DPD.
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
17
Table 1 MS data of ETI; degradation products A, B, C, D and the major fragment ions with their proposed structures. Name
Observed mass [M + 1]
Proposed formula
Calculated mass [M + 1]
Proposed structure
Relative error ×100
Etifoxine A B C
301.1121 301.1096 273.0616 230.0716
C17 H18 ClN2 O C17 H18 ClN2 O C15 H13 ClN2 O C14 H13 ClN
301.1108 301.1108 273.0716 230.0736
See Fig. 3 See Fig. 3 See Fig. 3 See Fig. 3
4.3175E−4 −3.9852E−4 −0.0037 −8.6929E−4
D
317.1033
C17 H18 ClN2 O2
317.1057
See Fig. 3
−7.5685E−4 Relative error ×100
Fragment
Observed mass (m/z)
Proposed formula
Calculated mass (m/z)
152
152.0687
C8 H7 NCl+
152.0267
0.0276
230
230.0749
C14 H13 NCl+
230.0736
5.6503E−4
256
256.0481
C15 H11 NOCl+
256.0607
−0.0049
chemical shifts in Table 2 with referring to Fig. 3 for the proton assignation number. If the proposed DPA structure is correct, the fundamental difference between DPA and ETI is given by the protons numbered 13 and 9. The proton 13 does not exist in ETI and the proton 9 is aliphatic in the ETI, ethylenic in DPA. ETI and DPA spectra (Fig. 4) and chemical shifts data (Table 2) confirm these differences. The chemical shifts of the DPA protons 13 and 10 are characteristic of an ureido group which appears with the opening of ETI oxazine ring. In Table 2, the chemical shifts of proton numbered 9 in ETI and DPA correspond to three ETI aliphatic protons and two DPA ethylenic protons respectively. This observation confirms again the opening of the oxazine ring present in ETI. These conclusions are supported by ETI and DPA 13 C NMR results presented in Fig. 4 and Table 2. Their 13 C NMR spectra differ principally by the carbons noted g and h (refer to Fig. 3 for carbon assignation). In ETI, g is in the oxazine cyle of the molecule, h is a primary carbon; their corresponding chemical shifts are 77.02 and 27.05 ppm respectively. In DPA, the chemical shifts of g and h move to 145.22 and 110.76 ppm; characteristic values of ethylenic carbons. Moreover, J-modulation applied to 13 C NMR of ETI and DPA reveals that the carbon h is directed upward for DPA (CH2 group) and downward for ETI (CH3 group). These results confirm the proposed structure for DPA. DPB appears at 13 min with all stress except with heat. Its mass spectra appear in Fig. 3 with pseudomolecular ion peak [M + H]+ at 273.0616 with the best molecular formula C15 H13 ClN2 O. Its proposed structure is given in Fig. 3 with 6-chloro-4-methyl-4phenyl-4-3,1-benzoxazin-2-amine is proposed as chemical name.
Proposed structure
Fig. 4 and Table 2 present DPB 1 H NMR spectrum and the corresponding proton chemical shifts (see Fig. 3 for the proton number). All chemical shifts indicate the structural formula proposed for DPB with 1.16 and 4.96 ppm for the protons 9 (in alkyl radical) and 10 (in amine radical) respectively. In the 13 C spectrum of B, the characteristic carbons are again g and h with a similar chemical shifts found in ETI for these carbons (see Fig. 3 for the letters). In DPB, the oxazine cycle is not opened as in ETI. J-modulation applied to 13 C NMR of DPB shows that the carbon h is directed downward (CH3 group). Taking account of all these results, the proposed structure for DPB is correct. Basic and oxidative degradations reveal DPC with 5 min retention time. Fig. 3 shows its mass spectrum. Pseudomolecular ion of C, [M + H]+ = 230.0716, its best molecular formula C14 H12 ClN allows to propose the structure in Fig. 3 and 4-chloro-2-(1phenylethenyl)aniline as molecular name. 1 H NMR spectrum of DPC given in Fig. 4 reveals two characteristics protons numbering 9 and 10 with a chemical shift of 5.25 and 3.44 ppm respectively. The first value (5.25 ppm) is specific to the protons present in CH2 group; the second (3.44 ppm) characterizes a primary amino group. These results confirm the proposed DPC formula and the chemical shifts of other protons comply with this chemical structure. In the 13 C spectrum of DPC (see Fig. 4), chemical shifts of g and h carbons are close to those found for DPA, they are characteristic of ethylenic carbons obtained after opening the oxazine cycle. In this case, J-modulation applied to 13 C NMR of DPB shows that the carbon h is directed upward (CH2 group). Considering the proposed structure, DPC probably originates also from DPA degradation.
18
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Table 2 1 H and 13 C chemical shifts in ETI, DPA, DPB, DPC and DPD (see Fig. 3 for the proton number and carbon assignments). 1
H RMN Results
H number ı (ppm)
ETI DPA DPB DPC DPD
H number ı (ppm)
13
1
2
3
4
5
6
7
6.78 7.33 6.64 6.92 6.92
7.31 7.33 7.28 7.31 7.40
7.76 7.71 7.30 7.36 7.54
7.09 7.17 7.14 7.15 7.22
7.09 7.17 7.14 7.15 7.22
7.09 7.17 7.14 7.15 7.22
7.09 7.17 7.14 7.15 7.22
8
9
ETI DPA DPB DPC
7.09 7.17 7.14 7.15
1.01 5.45 1.16 5.25
DPD
7.22
1.19
9 5.29 5.25
10
11
12
13
5.32 5.19 4.96 3.44
2.94 3.42
0.93 0.81
6.43
5.74
3.20
0.78
C RMN Results
c letter ı (ppm)
a
b
c
d
e
f
g
h
i
ETI DPA DPB DPC
123.65 123.34 121.99 122.37
126.42 126.11 126.97 127.08
127.16 123.22 123.79 122.92
128.98 128.66 129.11 128.06
122.84 127.91 128.34 128.41
143.50 142.77 143.11 143.75
77.02 145.22 77.48 144.98
27.05 110.76 27.11 111.39
140.50 142.01 142.31 141.49
DPD
123.87
127.94
124.02
128.77
128.11
143.47
78.03
27.39
141.33
c letter ı (ppm)
ETI DPA DPB DPC DPD
j
k
l
m
n
o
p
q
128.98 129.12 129.06 129.14 128.81
126.49 126.33 126.22 126.39 126.11
126.66 127.03 126.83 126.93 126.88
126.49 126.33 126.22 126.39 126.59
128.71 128.44 129.06 128.98 128.44
156.69 150.76 157.01
36.94 30.04
14.76 14.12
156.96
37.01
13.89
DPD results from the oxidative degradation only. The [M + H]+ at 317.1033 (Fig. 3) corresponds to [M + H]+ of ETI (301) + 16 which is the oxygen atom mass. Then, the proposed structure of DPD corresponds to the fixation of an oxygen atom by ETI during the H2 O2 action on nitrogen. Its proposed molecular name is 6-chloro-N-ethyl-N-hydroxy-4-methyl-4-phenyl-4H-3,1benzoxazin-2-amine; its structure is in Fig. 3. In our DPD proposition structure, NH group gets converted into NOH. This amine transformation to hydroxylamine was also found in the literature [13,14,26–28]. Considering DPD 1 H NMR spectrum (Fig. 4 and Table 2), all chemical shifts found allow to confirm the proposed DPD structure with the proton 10 (5.74 ppm) fixed on the NOH substituent. In DPD, we note that the proton 10 shows shielding behavior as compared to the ETI (5.74 and 5.32 ppm respectively). In the 1,3-oxazin-2-amine compounds, the hydrogen atom beared on the amino group is rather acidic due to the stability of the conjugated base enhanced by the negative charge delocalization. The pKa of the amino and hydroxylamino derivatives are similar, this induces very similar chemical shifts in the NMR spectrum for these two different hydrogens. The comparison of 13 C NMR spectra of DPD and ETI (see Fig. 4) does not show any remarkable differences even carbons o and p that surround the substituent NOH. J-modulation applied to 13 C NMR of DPD shows that the carbon h is directed downward (CH3 group). Table 2 resumes all the data, giving (i) structure of the major fragments found in ETI and DPA, DPB, DPC and DPDs spectra; (ii) relative errors between found and calculated mass of ETI; (iii) DPs and fragments. The DPs of ETI obtained under different stress actions are separated by HPLC, identified by LC-MS/TOF, 1 H and 13 C NMR spectroscopy. Taking account of all these data, the degradation pathway of ETI is proposed in Fig. 5 with the structures of the four DPs found and the principal fragments appearing in their MS spectra.
3.4. Validation The LC method is validated according to ICH guidelines [29]. Table 3 summarizes validation results. The specificity is determined by subjecting ETI to stress under various conditions. Using the selected chromatographic conditions, all the stress DPs are well separated from the ETI; no interferences are observed with the presence of excipients. The method is found to be specific. LOD and LOQ of ETI and its four DPA, DPB, DPC and DPDs estimated at a signal-to-noise ratio of 3 and 10, respectively are given in Table 3. In the range (10–30 g mL−1 ), the ETI response is linear (see Table 3 for correlation coefficient). The residual sum square found is 0.0088; the residuals are distributed at random around the regression line with no unidirectional tendency. To prove that the straight intercept detected is not significantly different from zero, a Student’s t-test is applied. The result obtained (t = 1.836) is lower than the theoretical value (t (0.05; 25) = 2.060). For the range specified Table 3 Validation parameters of the proposed method for the determination of ETI and its impurities A, B, C and D. Parameters
ETI
A
B
C
D
Slope Intercept Correlation coefficient Repeatability (RSD) Intermediate precision (RSD) Accuracy (%) LOD g mL−1 LOQ g mL−1
289,902 166,247 0.996
227,702 153,015 0.991
266,249 160,987 0.990
218,762 158,113 0.992
280,981 164,543 0.990
0.78
1.22
1.88
2.25
1.77
0.24
2.44
2.88
2.76
3.89
99.6 0.03 0.09
98.4 0.02 0.07
97.2 0.03 0.08
95.9 0.02 0.07
97.1 0.02 0.07
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
19
Fig. 5. Fragmentation pathways of ETI.
in experimental section, data for the four DPs given in Table 3 illustrate a good linearity curves for the four DPs. From repeatability, intermediate precision and accuracy data presented in Table 3, it appears clearly that the proposed indicatingstability method is precise and accurate considering ETI and its four DPs.
4. Conclusion In this paper, we present and validate a stability-indicating isocratic HPLC method for the determination of ETI. The drug is submitted to different degradation conditions (acidic, basic, oxidative, thermal and UV light stress). A number of DPs, depending
20
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
on the stress, appear in the chromatograms. The drug is found to be strongly degraded under thermal condition showing DPA only and less degraded under oxidative stress with the appearance of four compounds (DPA, DPB, DPC and DPD). The basic stress leads to the formation of DPA, DPB and DPC; the acid and photolytic effects on ETI produce DPA and DPB. The structures of the DPs are established by LC-MS/TOF and the corresponding ETI degradation pathway given. The proposed structures of the four DPs are confirmed by 1 H and 13 C NMR. The isocratic selected HPLC method is validated and is specific, linear, precise and accurate. Acknowledgements The authors would to thank the MS team of “Institut de chimie moleculaire”, University Paris XI, Orsay for their help in mass spectrometry and NMR experimentations. References [1] H.J. Kruse, H. Kuch, Etifoxine: evaluation of its anticonvulsant profile in mice in comparison with sodium valproate, phenytoin and clobazam, ArzneimittelForsch 35 (1985) 133–135. [2] R. Schlichter, V. Rybalchenko, P. Poisbeau, M. Verleye, J. Gillardin, Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine, Neuropharmacology 39 (2010) 1523–1535. [3] K.W. Gee, M.B. Tran, B. Minhtam, D.J. Hogenkamp, T.B. Johnstone, R.E. Bagnera, R.F. Yoshimura, J.C. Huang, J.D. Belluzzi, E.R. Whittemore, Limiting activity at 1-subunit-containing GABAA receptor subtypes reduces ataxia, J. Pharmacol. Exp. Ther. 332 (2010) 1040–1053. [4] M. Verleye, S. Dumas, I. Heulard, N. Krafft, J.M. Gillardin, Differential effects of etifoxine on anxiety-like behaviour and convulsions in BALB/cByJ and C57BL/6 J mice: any relation to overexpression of central GABAA receptor -2 subunits, Eur. Neuropsychopharm. 21 (2011) 457–470. [5] M. Bourin, M. Hascoet, Implication of 5-HT2 receptor subtypes in the mechanism of action of the GABAergic compound etifoxine in the four-plate test in Swiss mice, Behav. Brain Res. 208 (2010) 352–358. [6] M. Verleye, I. Heulard, J.M. Gillardin, The anxiolytic etifoxine protects against convulsant and anxiogenic aspects of the alcohol withdrawal syndrome in mice, Alcohol 43 (2009) 197–206. [7] N. Nguyen, E. Fakra, V. Pradel, E. Jouve, C. Alquier, M.E. Le Guern, J. Micallef, O. Blin, Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a doubleblind controlled study in general practice, Hum. Psychopharm. Clin. 21 (2006) 139–149. [8] J. Pang, H. Li, L. Li, Determination of etifoxine hydrochloride in etifoxine hydrochloride capsules, Zhongguo Yaoxue Zazhi (Beijing, China) 43 (2008) 1823–1826. [9] F.L. Sauvage, N. Picard, F. Saint-Marcoux, J.M. Gaulier, G. Lachâtre, P. Marquet, General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: applications and constraints, J. Sep. Sci. 32 (2009) 3074–3083. [10] ICH, Impurities in new drug substances, Q3A (R2), in: International Conference on Harmonisation, IFPMA, Geneva, 2006.
[11] A.F.B. Fauzee, R.B. Walker, Forced degradation studies of clobetasol 17propionate in methanol, propylene glycol, as bulk drug and cream formulations by RP-HPLC, J. Sep. Sci. 36 (2013) 849–856. [12] Q. Dong, J. Zhu, Q. Sui, C. Tang, X. Wang, Y. Yu, Optimization of mobile phase for the determination of Esomeprazole and related compounds and investigation of stress degradation by LC–MS, J. Sep. Sci. 36 (2013) 1200–1208. [13] T. Ramesh, P.N. Rao, R.N. Rao, LC–MS/MS method for the characterization of the forced degradation products of Entecavir, J. Sep. Sci. 37 (2014) 368–375. [14] A. Khedra, I. Darwish, F. Bamane, Analysis of abiraterone stress degradation behavior using liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry, J. Pharm. Biomed. Anal. 74 (2013) 77–82. [15] R.N. Rao, R.M. Vali, S.S. Raju, Liquid chromatography tandem mass spectrometric studies of indinavir sulphate and its forced degradation products, J. Pharm. Biomed. Anal. 74 (2013) 101–110. [16] M. Jamrogiewicz, B. Wielgomas, Detection of some volatile degradation products released during photoexposition of ranitidine in a solid state, J. Pharm. Biomed. Anal. 76 (2013) 177–182. [17] T.K. Thiyagarajan, B.M. Rao, P. Vukkum, J. Abdul Nasser, A. Singh, G.R. Deshpande, Chromatographia 68 (2008) 919–926. [18] M.R. Elghobashy, I. Lories, I.L. Bebawy, S.S. Abbas, R.F. Shokry, Stabilityindicating HPLC RP-TLC determination of Cefpirome sulfate with kinetic study, Chromatographia 76 (2013) 1141–1151. [19] L. Havlıkova, A. Pannyova, L. Matysova, P. Solich, Development of novel stability-indicating method for the determination of dimethindene maleate and its impurities, Chromatographia 76 (2013) 1545–1551. [20] R.N. Rao, H. Farah, K.P. Sahu, M. Janarthan, C.G. Naidu, Development and validation of a stability-indicating assay including the isolation and characterization of degradation products of metaxalone by LC-MS, Biomed. Chromatogr. 27 (2013) 1733–1740. [21] K.S. Rugani, R.N.S. Herida, Stability-indicating LC method for the determination of cephalothin in lyophilized powder for injection, Anal. Methods 6 (2014) 4437–4445. [22] R.N. Rao, B. Ramachandra, B. Sravan, Sara Khalid, LC–MS/MS structural characterization of stress degradation products including the development of a stability indicating assay of Darunavir: an anti-HIV drug, J. Pharm. Biomed. Anal. 89 (2014) 28–33. [23] M. Narayanam, T. Handa, P. Sharma, S. Jhajra, P.K. Muthe, P.K. Dappili, P.P. Ravi, R.P. Shah, S. Singh, Critical practical aspects in the application of liquid chromatography–mass spectrometric studies for the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 87 (2014) 191– 217. [24] ICH, International Conference on Harmonization Guideline on Stability Testing of New Drug Substances and Products, Q1A (R2), 2003. [25] CDER (Center for Drug Evaluation and Research) Reviewer Guidance, Validation of Chromatographic Methods, 1994. [26] P.H. Morgan, A.H. Beckett, Synthesis of some N-oxygenated products of 3,4dimethoxyampetamine and its N-alkyl derivatives, Tetrahedron 31 (1975) 2595–2601. [27] G. Zinner, W. Kliegel, On the oxidation of secondary amines with hydrogen peroxide. Hydroxylamine derivatives, Arch. Pharm. Ber. Dtsch. Pharm. Ges. 299 (1966) 166–173. [28] H. Beckett, S. Al-Sarraj, N-oxidation of primary and secondary amines to give hydroxylamines—a general metabolic route, J. Pharm. Pharmacol. 24 (1972) 916–917. [29] ICH, International Conference on Harmonization Guideline on Validation of Analytical Procedure, Test and Methodology, Geneva, Q2 (R1), 2005.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development of a stability-indicating HPLC method of Etifoxine with characterization of degradation products by LC-MS/TOF, 1 H and 13 C NMR Nadia Djabrouhou, Moulay-Hassane Guermouche ∗ Laboratoire de chromatographie, Faculté de chimie, USTHB, BP 32, El-Alia, Bab-Ezzouar, 16133 Alger, Algeria
a r t i c l e
i n f o
Article history: Received 10 May 2014 Received in revised form 18 July 2014 Accepted 20 July 2014 Available online 27 July 2014 Keywords: HPLC Etifoxine Stress degradation LC-MS/TOF 1 H and 13 C NMR Validation
a b s t r a c t This paper describes a new LC-MS/TOF method for the degradation products determination when Etifoxine (ETI) is submitted to different stress conditions. Chromatography is performed by using Kromasil C18 column (250 mm × 4.6 mm, 5 m particle size). The selected mobile phase consists of formate buffer 0.02 M, pH 3 and methanol (70/30, v/v). ETI is submitted to oxidative, acidic, basic, hydrolytic, thermal and UV light degradations. Detection is made at 254 nm by photodiode array detector and mass spectrometry. A number of degradation products (DPs) called DPA, DPB, DPC and DPD are found depending on the stress; DPA with heat, DPA and DPB in acidic media or under UV-light; DPA, DPB and DPC under basic stress; DPA, DPB, DPC and DPD with oxidation. LC-MS/TOF is used to characterize the four DPs of ETI resulting from different stress conditions. 1 H and 13 C NMR are used to confirm the DP structures. The ETI fragmentation pathway is proposed. The method is validated with reference to International Conference on Harmonization guidelines and ETI are selectively determined in presence of its DPs, demonstrating its stability-indicating nature. Finally, for the validation step, specificity, linearity, accuracy and precision are determined for ETI and its DPs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Etifoxine (ETI), is chemically designated as 6-chloro-N-ethyl4-methyl-4-phenyl-4H-benzo[d][1,3]oxazin-2-amine. It is used as an anxiolytic and anticonvulsant drug [1]. It has similar effects to benzodiazepine drugs with different side effects [2,3]. Differential effects of Etifoxine on anxiety-like behavior and convulsions in mice have been recently described in literature [4]. In another work, the authors demonstrate the role of 5-HT2 receptor subtypes in the mechanism of action of the GABAergic compound Etifoxine in mice [5]. The anxiolytic ETI protects against convulsant and anxiogenic aspects of the alcohol withdrawal syndrome are also studied [6]. Some authors investigate the impact of ETI on lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety [7]. A number of studies have been conducted and reported in recent years on the biological and pharmacological action of ETI. However, to the best of our knowledge, very few research papers tackle the problem of quantitative analysis of ETI. The unique relevant paper (in Chinese) on ETI analysis in
∗ Corresponding author. Tel.: +213 21247311; fax: +213 21247311. E-mail address: [email protected] (M.-H. Guermouche). http://dx.doi.org/10.1016/j.jpba.2014.07.017 0731-7085/© 2014 Elsevier B.V. All rights reserved.
pharmaceuticals that we identified presents a non-validated method which combines an Agilent TC-C18 column with mobile phase constituted with methanol–water (70:30, v/v) [8]. Another general work on screening procedure for the characterization of human drug metabolites briefly cited the case of Etifoxine [9]. Therefore, it is imperative to develop a rapid and simple procedure of ETI HPLC analysis. In recent years, it is strongly recommended that any HPLC development method of active pharmaceutical ingredients should include a good peak separation between a drug and its DPs; official methodology exists [10]. A great number of papers describe this type of studies for several drugs using HPLC or LC-MS-MS. The stress degradation are performed on Clobetasol 17-propionate [11], Esomeprazole [12], Entecavir [13], Abiraterone [14], Indinavir sulphate [15], Ranitidine [16], Desipramine hydrochloride [17], Cefpirome [18] and Dimethindene Maleate [19]. More recently, stress degradations of Metaxalone [20], cephalothin [21] and Mometsone Furoate [22] are studied. Moreover, recent study proposes a critical review of the HPLC-MS use in this field [23]. To the best of our knowledge, there is no stability-indicating method of ETI. The ultimate objective of the present work is to present a new and validated stability-indicating HPLC method for the quantitative determination of ETI in capsules involving an
12
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
acidic, basic, oxidative, thermal and light degradation of ETI. The chemical structures of the different DPs produced by stress are investigated by LC-MS/TOF and 1 H and 13 C NMR.
tailing factor, resolution between ETI and DPs) are measured for each mobile phase. 2.4. Stress studies
2. Experimental 2.1. Reagents Etifoxine hydrochloride (99.3%) is kindly provided by the National Laboratory Control of Pharmaceutical Products (Algiers, Algeria). The substance is used as received for preparing standard solutions. Ammonium formate is obtained from Prolabo (France), formic acid and LC quality methanol are purchased from Sigma Aldrich (USA). 2.2. Instrumentation For the LC-MS/TOF experiments, Ultimate 3000 chromatograph (Dionex, USA) coupled to MicroTof Q II (Brüker Daltonics, Germany) is used. Chromatography is performed on the same conditions selected in LC experimentation. The following LC-MS/TOF parameters are used: source type, ESI; ion polarity, positive; end plate offset voltage, −500 V; capillary voltage, 4000 V; nebulizer pressure, 69.6 psi, dry gas, 12.0 L min−1 ; dry heater, 300 ◦ C; collision cell RF, 150.0 Vpp; scan begin 50 m/z; scan end, 600 m/z. Data are collected on Compass (Brüker Daltonics, Germany) software. LC analysis is performed with a Waters chromatograph equipped with a 600E pump, 7725i Rheodyne injector with 20 L sample loop, photodiode array detector. ETI is eluted on a KROMASIL C18 analytical column (250 mm × 4.6 mm i.d., 5 m particles) from AKZO Nobel (Sweden) under reversed phase partition chromatographic conditions. The flow rate is 1.0 mL min−1 and the analyte is monitored at the maximal absorption of ETI (254 nm). Data are collected with Empower program (Waters). Before the analysis, the mobile phase is degassed by using a Branson sonicator (Branson Ultrasonics, USA) and filtered through a 0.45 m filter. The system is equilibrated before each injection. NMR experiments are performed using a 400-MHz Brüker NMR Spectrometer, with TMS as a reference and CDCl3 as solvent of ETI and DPs samples. 2.3. Determination of the optimal HPLC conditions Several trials are carried out to obtain a good resolution between the drug and its DPs. These trials involved the use of different mobile phases with different ratios, and different pH. The suitable mobile phase should control the selectivity and achieve reproducible separations with an acceptable peak shape. It is prepared daily and filtered through a 0.45 m Millipore filter and ultrasonicated for 30 min before use. Ammonium acetate has a pKa of 4.8 and its buffer range (pKa ±1 unit) is 3.8 and 5.8. Therefore, the mobile phases are made of methanol (volumetric composition varied from 65 to 75%) and formate buffer 0.02 M (pH from 3 to 4). The use of methanol was important to increase the retention factor and to obtain a better resolution of drug and DPs. Four mobile phases are tested: Mobile phase 1: formate buffer 0.02 M (pH 4)–MeOH, 35–65, v/v. Mobile phase 2: formate buffer 0.02 M (pH 3)–MeOH, 30–70, v/v. Mobile phase 3: formate buffer 0.02 M (pH 3)–MeOH, 25–75, v/v. Mobile phase 4: formate buffer 0.02 M (pH 3.5)–MeOH, 25–75, v/v. Flow rate is fixed to 1 mL min−1 . Injections are made in triplicate. Chromatographic parameters (retention time, plate number and
The stability of ETI under the conditions of hydrolysis, oxidation and thermal degradation are investigated as mentioned in International Conference on harmonization [24]. In each case, the stress time is maintained long enough to achieve at least 20% degradation of the drug initial amount. The stress experiments are made with an initial solution containing ETI at 2 mg mL−1 of ETI in methanol except for thermal and photolytic degradation. Acidic degradation is carried out by adding 1 mL of 2 M HCl to 1 mL of the initial condition; 20% of ETI is degraded after 60 h. Alkali degradation is performed by mixing 1 mL of initial solution and 1 mL of 2 M NaOH; 120 h are necessary to obtain 20% of degadation. 20% of oxidative degradation is obtained by exposing 1 mL of initial solution to 1 mL of 3% (v/v) H2 O2 during 60 h. Twenty percent of ETI degradation with heat was performed by heating pure ETI at 80 ◦ C during 4 h. Light degradation is obtained by submitting ETI to a light from Mercury lamp (50 W Mercury high pressure lamp, luminous flux 2000 Im) during 24 h. Before LC/MS, solutions resulting from stress are diluted 50 times with mobile phase, filtered under 25 mm Millex-HV membrane syringe filter (pore size 0.25 m). The samples of ETI degraded under different conditions are isolated by semi-preparative HPLC using Kromasil C18 (250 mm × 10 mm, 5 m) column with the same mobile phase selected after the determination of the optimal chromatographic conditions. Injection volume of the sample is fixed to 200 L. The column eluents are monitored by a photo diode array detector at 254 nm. Four collected solutions containing DPA, DPB, DPC and DPD respectively are extracted with 20 mL of chloroform, evaporated to dryness. The residue of each solution with one DP is collected and used for the validation assays and NMR experiments. 2.5. Validation of the HPLC method Specificity of the developed LC method for ETI is examined in the presence of its degradation products (namely DPA, DPB, DPC and DPD) and the excipients present in capsules (lactose monohydrate, talc, microcrystalline cellulose, colloidal anhydrous silica and magnesium stearate). A placebo capsule matrix solution is prepared from a weighted mixture of the excipients without the drug respecting their weight composition in the commercial capsules. It is mixed with oxidative stress sample solution to improve specificity because hydrogen peroxide action on ETI generates four DPs. The limit of detection (LOD) and quantification (LOQ) for ETI and its four DPs are determined at a signal-to-noise ratio of 3 and 10, respectively. Linearity is carried out with external standard method for ETI and its DPs. Five standard solutions of ETI (10, 15, 20, 25 and 30 g mL−1 ) are prepared separately in methanol. These solutions are made daily and analyzed immediately after preparation. They correspond to 50%, 75%, 100%, 125% and 150% of ETI. For each DP, the linearity is examined from 0.5 to 4 g mL−1 (4 g mL−1 corresponds to 20% of ETI degradation). All solutions are filtered using a 25 mm Millex-HV membrane syringe filter (pore size 0.45 m). Twenty microliter of each solution is injected in triplicates. The peak area of ETI and DPs are measured, a calibration curves are obtained from the least-squares linear regression of the identified peak area. Precision is evaluated as the repeatability and intermediate precision levels. Six samples (100% level: 20 g mL−1 for ETI and 4 g mL−1 for each DP) are freshly prepared and triplicates
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
13
Fig. 1. (a) Bar graphs showing the variation of: (a) ETI chromatographic parameters with methanol composition and pH; (b) resolution between DPs and ETI with methanol composition and pH.
measurements are used to perform repeatability. Intermediate precision is calculated from data of six independent standards with a level of 100% (20 g mL−1 for ETI and 4 g mL−1 for each DP). These standards are analyzed by two independent analysts, in triplicates, during three different days. Accuracy is measured at concentration levels of 50% (three samples), 100% (three samples) and 150% (three samples) where a known amount of the active is added to an amount of placebo. Accuracy is estimated with ETI or degradation product RSD%.
3. Results and discussion 3.1. Determination of the optimal chromatographic conditions All the results are given in Fig. 1a. To select the optimal chromatographic conditions, plate number effect is examined first, tailing factor of ETI peak is the second parameter considered. The mobile phase made of formate buffer 0.02 M (pH 3)–MeOH, 30–70, v/v gives the greatest plate number (N = 5081), the lowest tailing factor value (1.06). The corresponding retention time is convenient (retention time = 7.5 min). On the other hand, using the different tested mobile phases, resolutions between ETI and the DPs given by the stressed sample solution resulting from oxidative stress are calculated and plotted in Fig. 1b. With all the mobile phases, elution order is DPC, ETI and DPD, DPB and finally DPA. The best resolution between DPC and ETI is given by the mobile phase 1 but the DPA retention time exceeds 43 min. When we consider the resolutions between ETI and DPD, DPD and DPB, DPB and DPA, the greatest values are given by the mobile phase 2. Moreover, with this solvent, the A retention time is convenient and around 20 min. Finally, we select the mobile phase 1 made of 70% of methanol in formate buffer 0.02 M (v/v) adjusted to pH 3. Suitability parameters found including plate number, tailing factor and retention factor, comply with the FDA guidelines on Validation of Chromatographic Methods [25].
3.2. Forced degradation studies 3.2.1. Stress degradation by hydrolysis under acidic conditions In the conditions described in Section 2, 20% degradation occurs after about 70 h. The corresponding LC chromatogram is given in
Fig. 2a. Two DPs called DPA and DPB are observed at 21 and 13 min respectively. 3.2.2. Stress degradation by hydrolysis under basic conditions It seems that ETI degradation is slower in basic medium compared to acidic solution. In basic solution, with the conditions described in the experimental part, 20% of ETI degradation is not reached after 120 h of stress. Fig. 2b illustrates the corresponding chromatogram. We observe three DPs; two of them have the same retention times of DPA and DPB found under the acidic attack. The third DP called DPC has 4 min as retention time. 3.2.3. Oxidative stress ETI is found to be more sensitive to stress with 3% H2 O2 than to acidic or basic media. About 45 h of exposure are required to obtain 20% degradation with 3% H2 O2 . Fig. 2c shows the corresponding chromatogram where four DPs are found. Three of them are already detected at the same times when ETI is submitted to acidic (DPA and DPB) or basic stress (DPA, DPB and DPC). The new degradation product called DPD has a 10 min retention time. 3.2.4. Thermal degradation The highest stress effect is found with the temperature action. After 2 h of heat at 80 ◦ C, 20% of ETI are degraded leading to the formation of only one DP having the same retention time of DPA. Chromatogram appearing in Fig. 2d is relative to this effect. 3.2.5. Degradation under UV light Compared to the other stress parameters, UV light effect on ETI is faster than acidic stress; it produces two DPs at the same retention time than DPA and DPB (Fig. 2e) and 20% of ETI are lost after 24 h. Fig. 2f shows UV spectra of DPA, DPB, DPC and DPD recorded by PDA detector. The DPs found with the other stress give the same spectra (DPA and DPB in acidic media and after photolytic action; DPA, DPB and DPC with basic stress; DPA after thermal degradation). As the DPs have the same retention times and the same UV spectra with all stresses, we can assume that the DP structures can be the same.
14
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Fig. 2. Chromatograms ETI stress degradation with HCl (a), NaOH (b), H2 O2 (c), heat (d) and photolytic action (e). UV spectra of ETI and the DPs A, B, C and D (f).
3.3. LC-MS/TOF, 1 H and 13 C NMR results LC-MS/TOF is used to identify the DPs resulted from ETI degradation. It is important to remember that under H2 O2 stress, ETI gives four degradation products DPA, DPB, DPC and DPD at approximately 21, 13, 4 and 10 min as retention times; ETI is eluted in about 7 min. Mass spectra of ETI, DPA, DPB, DPC and DPD are given in Fig. 3 in the rectangles reported as ETI, Degradation product A, Degradation product B, Degradation product C and Degradation product D respectively. In basic media, three DPs appear. They have the same retention times, UV spectra and mass spectra than the DPA, DPB and DPC found with oxidative stress. We can conclude that it is most likely the same products. The same reasoning can be held with
other stress; thermal attack gives DPA only while DPA and DPB are obtained with acidic or UV stress. The unique other DP is done by oxidative action. These conclusions must be confirmed by LCMS/TOF, 1 H and 13 C NMR. For ETI, DPA DPB DPC and DPD, mass spectra are given if Fig. 3 where hydrogens and carbons are identified by numbers and letters respectively. In Fig. 4, 1 H and 13 C NMR (including J-mode) spectra of ETI, DPA DPB DPC and DPD are presented. Their corresponding chemical shifts are given in Table 1. ETI mass spectrum of DPA is given in Fig. 3. According to mass spectrum formula report, the ion with 301 molecular weight corresponds to a proposed formula C17 H18 ClN2 O equivalent protonated molecular ions [M + H]+ at m/z = 301.1121. ETI MS/TOF spectrum of [M + H]+ ions (m/z 301) eluting at approximately 7 min retention
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
15
Fig. 3. Mass spectra of ETI (ETI, rectangle), DPA (A, rectangle), DPB (B, rectangle), DPC (C, rectangle) and DPD (D, rectangle). Numbers and letters assigned to H and C atoms present in ETI, DPA, DPB, DPC and DPD.
time displays product ions at m/z 256.0481 and 230.0749 Da; their best proposed formula are C15 H11 NOCl+ and C14 H13 NCl+ respectively. Their proposed structures are given in Table 1. The 1 H NMR spectra of ETI and the corresponding proton chemical shifts are presented in Fig. 4 and Table 2 respectively. Mass spectrum of degradation product A (retention time about 21 min) appears with all stress with a pseudomolecular ion peak [M + H]+ at m/z 301.1096. It is surprising to note that ETI and DPA have the same molecular mass. Comparing the mass spectra of
ETI and DPA, It is important to note that the ETI fragment located at 256.0481 does not exist in DPA which suggests that probably, the oxazin-2-amine cycle does not exist in DPA, it undergoes an opening the oxazine ring present in ETI giving DPA with formation of carbonyl and ethylenic functions. DPA chemical name is 1-(5-chloro-2-(3-ethylureido) phenyl)-1-phenylethylene. Another fragment found (m/z = 230) is common to ETI and DPA. Its structure is given in Fig. 3 and must be confirmed by NMR. In Fig. 4, we present 1 H NMR and 13 C spectra of DPA and its corresponding
16
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Fig. 4.
1
H and 13 C NMR (including J-mode spectra) of ETI, DPA, DPPB, DPC and DPD.
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
17
Table 1 MS data of ETI; degradation products A, B, C, D and the major fragment ions with their proposed structures. Name
Observed mass [M + 1]
Proposed formula
Calculated mass [M + 1]
Proposed structure
Relative error ×100
Etifoxine A B C
301.1121 301.1096 273.0616 230.0716
C17 H18 ClN2 O C17 H18 ClN2 O C15 H13 ClN2 O C14 H13 ClN
301.1108 301.1108 273.0716 230.0736
See Fig. 3 See Fig. 3 See Fig. 3 See Fig. 3
4.3175E−4 −3.9852E−4 −0.0037 −8.6929E−4
D
317.1033
C17 H18 ClN2 O2
317.1057
See Fig. 3
−7.5685E−4 Relative error ×100
Fragment
Observed mass (m/z)
Proposed formula
Calculated mass (m/z)
152
152.0687
C8 H7 NCl+
152.0267
0.0276
230
230.0749
C14 H13 NCl+
230.0736
5.6503E−4
256
256.0481
C15 H11 NOCl+
256.0607
−0.0049
chemical shifts in Table 2 with referring to Fig. 3 for the proton assignation number. If the proposed DPA structure is correct, the fundamental difference between DPA and ETI is given by the protons numbered 13 and 9. The proton 13 does not exist in ETI and the proton 9 is aliphatic in the ETI, ethylenic in DPA. ETI and DPA spectra (Fig. 4) and chemical shifts data (Table 2) confirm these differences. The chemical shifts of the DPA protons 13 and 10 are characteristic of an ureido group which appears with the opening of ETI oxazine ring. In Table 2, the chemical shifts of proton numbered 9 in ETI and DPA correspond to three ETI aliphatic protons and two DPA ethylenic protons respectively. This observation confirms again the opening of the oxazine ring present in ETI. These conclusions are supported by ETI and DPA 13 C NMR results presented in Fig. 4 and Table 2. Their 13 C NMR spectra differ principally by the carbons noted g and h (refer to Fig. 3 for carbon assignation). In ETI, g is in the oxazine cyle of the molecule, h is a primary carbon; their corresponding chemical shifts are 77.02 and 27.05 ppm respectively. In DPA, the chemical shifts of g and h move to 145.22 and 110.76 ppm; characteristic values of ethylenic carbons. Moreover, J-modulation applied to 13 C NMR of ETI and DPA reveals that the carbon h is directed upward for DPA (CH2 group) and downward for ETI (CH3 group). These results confirm the proposed structure for DPA. DPB appears at 13 min with all stress except with heat. Its mass spectra appear in Fig. 3 with pseudomolecular ion peak [M + H]+ at 273.0616 with the best molecular formula C15 H13 ClN2 O. Its proposed structure is given in Fig. 3 with 6-chloro-4-methyl-4phenyl-4-3,1-benzoxazin-2-amine is proposed as chemical name.
Proposed structure
Fig. 4 and Table 2 present DPB 1 H NMR spectrum and the corresponding proton chemical shifts (see Fig. 3 for the proton number). All chemical shifts indicate the structural formula proposed for DPB with 1.16 and 4.96 ppm for the protons 9 (in alkyl radical) and 10 (in amine radical) respectively. In the 13 C spectrum of B, the characteristic carbons are again g and h with a similar chemical shifts found in ETI for these carbons (see Fig. 3 for the letters). In DPB, the oxazine cycle is not opened as in ETI. J-modulation applied to 13 C NMR of DPB shows that the carbon h is directed downward (CH3 group). Taking account of all these results, the proposed structure for DPB is correct. Basic and oxidative degradations reveal DPC with 5 min retention time. Fig. 3 shows its mass spectrum. Pseudomolecular ion of C, [M + H]+ = 230.0716, its best molecular formula C14 H12 ClN allows to propose the structure in Fig. 3 and 4-chloro-2-(1phenylethenyl)aniline as molecular name. 1 H NMR spectrum of DPC given in Fig. 4 reveals two characteristics protons numbering 9 and 10 with a chemical shift of 5.25 and 3.44 ppm respectively. The first value (5.25 ppm) is specific to the protons present in CH2 group; the second (3.44 ppm) characterizes a primary amino group. These results confirm the proposed DPC formula and the chemical shifts of other protons comply with this chemical structure. In the 13 C spectrum of DPC (see Fig. 4), chemical shifts of g and h carbons are close to those found for DPA, they are characteristic of ethylenic carbons obtained after opening the oxazine cycle. In this case, J-modulation applied to 13 C NMR of DPB shows that the carbon h is directed upward (CH2 group). Considering the proposed structure, DPC probably originates also from DPA degradation.
18
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
Table 2 1 H and 13 C chemical shifts in ETI, DPA, DPB, DPC and DPD (see Fig. 3 for the proton number and carbon assignments). 1
H RMN Results
H number ı (ppm)
ETI DPA DPB DPC DPD
H number ı (ppm)
13
1
2
3
4
5
6
7
6.78 7.33 6.64 6.92 6.92
7.31 7.33 7.28 7.31 7.40
7.76 7.71 7.30 7.36 7.54
7.09 7.17 7.14 7.15 7.22
7.09 7.17 7.14 7.15 7.22
7.09 7.17 7.14 7.15 7.22
7.09 7.17 7.14 7.15 7.22
8
9
ETI DPA DPB DPC
7.09 7.17 7.14 7.15
1.01 5.45 1.16 5.25
DPD
7.22
1.19
9 5.29 5.25
10
11
12
13
5.32 5.19 4.96 3.44
2.94 3.42
0.93 0.81
6.43
5.74
3.20
0.78
C RMN Results
c letter ı (ppm)
a
b
c
d
e
f
g
h
i
ETI DPA DPB DPC
123.65 123.34 121.99 122.37
126.42 126.11 126.97 127.08
127.16 123.22 123.79 122.92
128.98 128.66 129.11 128.06
122.84 127.91 128.34 128.41
143.50 142.77 143.11 143.75
77.02 145.22 77.48 144.98
27.05 110.76 27.11 111.39
140.50 142.01 142.31 141.49
DPD
123.87
127.94
124.02
128.77
128.11
143.47
78.03
27.39
141.33
c letter ı (ppm)
ETI DPA DPB DPC DPD
j
k
l
m
n
o
p
q
128.98 129.12 129.06 129.14 128.81
126.49 126.33 126.22 126.39 126.11
126.66 127.03 126.83 126.93 126.88
126.49 126.33 126.22 126.39 126.59
128.71 128.44 129.06 128.98 128.44
156.69 150.76 157.01
36.94 30.04
14.76 14.12
156.96
37.01
13.89
DPD results from the oxidative degradation only. The [M + H]+ at 317.1033 (Fig. 3) corresponds to [M + H]+ of ETI (301) + 16 which is the oxygen atom mass. Then, the proposed structure of DPD corresponds to the fixation of an oxygen atom by ETI during the H2 O2 action on nitrogen. Its proposed molecular name is 6-chloro-N-ethyl-N-hydroxy-4-methyl-4-phenyl-4H-3,1benzoxazin-2-amine; its structure is in Fig. 3. In our DPD proposition structure, NH group gets converted into NOH. This amine transformation to hydroxylamine was also found in the literature [13,14,26–28]. Considering DPD 1 H NMR spectrum (Fig. 4 and Table 2), all chemical shifts found allow to confirm the proposed DPD structure with the proton 10 (5.74 ppm) fixed on the NOH substituent. In DPD, we note that the proton 10 shows shielding behavior as compared to the ETI (5.74 and 5.32 ppm respectively). In the 1,3-oxazin-2-amine compounds, the hydrogen atom beared on the amino group is rather acidic due to the stability of the conjugated base enhanced by the negative charge delocalization. The pKa of the amino and hydroxylamino derivatives are similar, this induces very similar chemical shifts in the NMR spectrum for these two different hydrogens. The comparison of 13 C NMR spectra of DPD and ETI (see Fig. 4) does not show any remarkable differences even carbons o and p that surround the substituent NOH. J-modulation applied to 13 C NMR of DPD shows that the carbon h is directed downward (CH3 group). Table 2 resumes all the data, giving (i) structure of the major fragments found in ETI and DPA, DPB, DPC and DPDs spectra; (ii) relative errors between found and calculated mass of ETI; (iii) DPs and fragments. The DPs of ETI obtained under different stress actions are separated by HPLC, identified by LC-MS/TOF, 1 H and 13 C NMR spectroscopy. Taking account of all these data, the degradation pathway of ETI is proposed in Fig. 5 with the structures of the four DPs found and the principal fragments appearing in their MS spectra.
3.4. Validation The LC method is validated according to ICH guidelines [29]. Table 3 summarizes validation results. The specificity is determined by subjecting ETI to stress under various conditions. Using the selected chromatographic conditions, all the stress DPs are well separated from the ETI; no interferences are observed with the presence of excipients. The method is found to be specific. LOD and LOQ of ETI and its four DPA, DPB, DPC and DPDs estimated at a signal-to-noise ratio of 3 and 10, respectively are given in Table 3. In the range (10–30 g mL−1 ), the ETI response is linear (see Table 3 for correlation coefficient). The residual sum square found is 0.0088; the residuals are distributed at random around the regression line with no unidirectional tendency. To prove that the straight intercept detected is not significantly different from zero, a Student’s t-test is applied. The result obtained (t = 1.836) is lower than the theoretical value (t (0.05; 25) = 2.060). For the range specified Table 3 Validation parameters of the proposed method for the determination of ETI and its impurities A, B, C and D. Parameters
ETI
A
B
C
D
Slope Intercept Correlation coefficient Repeatability (RSD) Intermediate precision (RSD) Accuracy (%) LOD g mL−1 LOQ g mL−1
289,902 166,247 0.996
227,702 153,015 0.991
266,249 160,987 0.990
218,762 158,113 0.992
280,981 164,543 0.990
0.78
1.22
1.88
2.25
1.77
0.24
2.44
2.88
2.76
3.89
99.6 0.03 0.09
98.4 0.02 0.07
97.2 0.03 0.08
95.9 0.02 0.07
97.1 0.02 0.07
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
19
Fig. 5. Fragmentation pathways of ETI.
in experimental section, data for the four DPs given in Table 3 illustrate a good linearity curves for the four DPs. From repeatability, intermediate precision and accuracy data presented in Table 3, it appears clearly that the proposed indicatingstability method is precise and accurate considering ETI and its four DPs.
4. Conclusion In this paper, we present and validate a stability-indicating isocratic HPLC method for the determination of ETI. The drug is submitted to different degradation conditions (acidic, basic, oxidative, thermal and UV light stress). A number of DPs, depending
20
N. Djabrouhou, M.-H. Guermouche / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 11–20
on the stress, appear in the chromatograms. The drug is found to be strongly degraded under thermal condition showing DPA only and less degraded under oxidative stress with the appearance of four compounds (DPA, DPB, DPC and DPD). The basic stress leads to the formation of DPA, DPB and DPC; the acid and photolytic effects on ETI produce DPA and DPB. The structures of the DPs are established by LC-MS/TOF and the corresponding ETI degradation pathway given. The proposed structures of the four DPs are confirmed by 1 H and 13 C NMR. The isocratic selected HPLC method is validated and is specific, linear, precise and accurate. Acknowledgements The authors would to thank the MS team of “Institut de chimie moleculaire”, University Paris XI, Orsay for their help in mass spectrometry and NMR experimentations. References [1] H.J. Kruse, H. Kuch, Etifoxine: evaluation of its anticonvulsant profile in mice in comparison with sodium valproate, phenytoin and clobazam, ArzneimittelForsch 35 (1985) 133–135. [2] R. Schlichter, V. Rybalchenko, P. Poisbeau, M. Verleye, J. Gillardin, Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine, Neuropharmacology 39 (2010) 1523–1535. [3] K.W. Gee, M.B. Tran, B. Minhtam, D.J. Hogenkamp, T.B. Johnstone, R.E. Bagnera, R.F. Yoshimura, J.C. Huang, J.D. Belluzzi, E.R. Whittemore, Limiting activity at 1-subunit-containing GABAA receptor subtypes reduces ataxia, J. Pharmacol. Exp. Ther. 332 (2010) 1040–1053. [4] M. Verleye, S. Dumas, I. Heulard, N. Krafft, J.M. Gillardin, Differential effects of etifoxine on anxiety-like behaviour and convulsions in BALB/cByJ and C57BL/6 J mice: any relation to overexpression of central GABAA receptor -2 subunits, Eur. Neuropsychopharm. 21 (2011) 457–470. [5] M. Bourin, M. Hascoet, Implication of 5-HT2 receptor subtypes in the mechanism of action of the GABAergic compound etifoxine in the four-plate test in Swiss mice, Behav. Brain Res. 208 (2010) 352–358. [6] M. Verleye, I. Heulard, J.M. Gillardin, The anxiolytic etifoxine protects against convulsant and anxiogenic aspects of the alcohol withdrawal syndrome in mice, Alcohol 43 (2009) 197–206. [7] N. Nguyen, E. Fakra, V. Pradel, E. Jouve, C. Alquier, M.E. Le Guern, J. Micallef, O. Blin, Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a doubleblind controlled study in general practice, Hum. Psychopharm. Clin. 21 (2006) 139–149. [8] J. Pang, H. Li, L. Li, Determination of etifoxine hydrochloride in etifoxine hydrochloride capsules, Zhongguo Yaoxue Zazhi (Beijing, China) 43 (2008) 1823–1826. [9] F.L. Sauvage, N. Picard, F. Saint-Marcoux, J.M. Gaulier, G. Lachâtre, P. Marquet, General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: applications and constraints, J. Sep. Sci. 32 (2009) 3074–3083. [10] ICH, Impurities in new drug substances, Q3A (R2), in: International Conference on Harmonisation, IFPMA, Geneva, 2006.
[11] A.F.B. Fauzee, R.B. Walker, Forced degradation studies of clobetasol 17propionate in methanol, propylene glycol, as bulk drug and cream formulations by RP-HPLC, J. Sep. Sci. 36 (2013) 849–856. [12] Q. Dong, J. Zhu, Q. Sui, C. Tang, X. Wang, Y. Yu, Optimization of mobile phase for the determination of Esomeprazole and related compounds and investigation of stress degradation by LC–MS, J. Sep. Sci. 36 (2013) 1200–1208. [13] T. Ramesh, P.N. Rao, R.N. Rao, LC–MS/MS method for the characterization of the forced degradation products of Entecavir, J. Sep. Sci. 37 (2014) 368–375. [14] A. Khedra, I. Darwish, F. Bamane, Analysis of abiraterone stress degradation behavior using liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry, J. Pharm. Biomed. Anal. 74 (2013) 77–82. [15] R.N. Rao, R.M. Vali, S.S. Raju, Liquid chromatography tandem mass spectrometric studies of indinavir sulphate and its forced degradation products, J. Pharm. Biomed. Anal. 74 (2013) 101–110. [16] M. Jamrogiewicz, B. Wielgomas, Detection of some volatile degradation products released during photoexposition of ranitidine in a solid state, J. Pharm. Biomed. Anal. 76 (2013) 177–182. [17] T.K. Thiyagarajan, B.M. Rao, P. Vukkum, J. Abdul Nasser, A. Singh, G.R. Deshpande, Chromatographia 68 (2008) 919–926. [18] M.R. Elghobashy, I. Lories, I.L. Bebawy, S.S. Abbas, R.F. Shokry, Stabilityindicating HPLC RP-TLC determination of Cefpirome sulfate with kinetic study, Chromatographia 76 (2013) 1141–1151. [19] L. Havlıkova, A. Pannyova, L. Matysova, P. Solich, Development of novel stability-indicating method for the determination of dimethindene maleate and its impurities, Chromatographia 76 (2013) 1545–1551. [20] R.N. Rao, H. Farah, K.P. Sahu, M. Janarthan, C.G. Naidu, Development and validation of a stability-indicating assay including the isolation and characterization of degradation products of metaxalone by LC-MS, Biomed. Chromatogr. 27 (2013) 1733–1740. [21] K.S. Rugani, R.N.S. Herida, Stability-indicating LC method for the determination of cephalothin in lyophilized powder for injection, Anal. Methods 6 (2014) 4437–4445. [22] R.N. Rao, B. Ramachandra, B. Sravan, Sara Khalid, LC–MS/MS structural characterization of stress degradation products including the development of a stability indicating assay of Darunavir: an anti-HIV drug, J. Pharm. Biomed. Anal. 89 (2014) 28–33. [23] M. Narayanam, T. Handa, P. Sharma, S. Jhajra, P.K. Muthe, P.K. Dappili, P.P. Ravi, R.P. Shah, S. Singh, Critical practical aspects in the application of liquid chromatography–mass spectrometric studies for the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 87 (2014) 191– 217. [24] ICH, International Conference on Harmonization Guideline on Stability Testing of New Drug Substances and Products, Q1A (R2), 2003. [25] CDER (Center for Drug Evaluation and Research) Reviewer Guidance, Validation of Chromatographic Methods, 1994. [26] P.H. Morgan, A.H. Beckett, Synthesis of some N-oxygenated products of 3,4dimethoxyampetamine and its N-alkyl derivatives, Tetrahedron 31 (1975) 2595–2601. [27] G. Zinner, W. Kliegel, On the oxidation of secondary amines with hydrogen peroxide. Hydroxylamine derivatives, Arch. Pharm. Ber. Dtsch. Pharm. Ges. 299 (1966) 166–173. [28] H. Beckett, S. Al-Sarraj, N-oxidation of primary and secondary amines to give hydroxylamines—a general metabolic route, J. Pharm. Pharmacol. 24 (1972) 916–917. [29] ICH, International Conference on Harmonization Guideline on Validation of Analytical Procedure, Test and Methodology, Geneva, Q2 (R1), 2005.
