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Stability-indicating RP-UPLC method for the simultaneous determination of potential degradation and process impurities of amlodipine basylate and benazepril HCl in pharmaceutical dosage form.

1. INTRODUCTION

High blood pressure can be treated with number of drugs depending upon the causes which are responsible for it. It is increasingly appreciated that the elusive goal of a normal blood pressure is achieved only if multi-drug therapy is employed [1].

Amlodipine, 2-[(2-aminoethoxy)methyl] -4-(2chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine dicarboxylic acid- 3-ethyl-5-methyl ester, a calcium channel blocker, is used alone or with benazepril, an angiotensin-converting enzyme inhibitor, to treat hypertension, chronic stable angina pectoris, and prinzmetal's variant angina [2].

Benazepril, 2-[(3S)-3-[[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino]-2-oxo-4, 5-dihydro-3H-1-benzazepin-1-yl]acetic acid is an angiotensinconverting enzyme (ACE) inhibitor, is a prodrug which, when hydrolyzed by esterases to its active Benazeprilat, is used to treat hypertension and heart failure, to reduce proteinuria and renal disease in patients with nephropathies, and to prevent stroke, myocardial infarction, and cardiac death in high-risk patients [3].

AB and BH are official in USP 32 as individual monographs [4]. Detailed literature survey for AB revealed that several analytical methods are reported for the determination of amlodipine and atorvastatine by HPLC [5-7], amlodipine and atenolol by HPLC [8] and by HPTLC [9], amlodipine and metaprolol by HPLC [10-11] and amlodipine alone by HPLC [12-16], HPTLC [17], LC-MS [18-25], GC [26], amperometric detection [27-29] spectophotometric detection [30-31], fluorescence detection [32-33], by voltammetry [34-35], and derivatization methods [36]. Similarly, literature survay for BH revealed that few techniques have 'Corresponding author. E-mail: mazahar 64@rediffmail.com been reported for the determination of benazepril hydrochloride alone by HPLC [37-39], HPTLC [40], CE [41-43] GC-MS [44-45], LC-MS [46-47], spectrophotometric detection [48-49] and by voltammetry [50] from pharmaceutical dosages form. Few methods have been reported for the simultaneous determination of AB and BH which describes the RPHPLC procedure [51] and the other describes an HPTLC procedure [52]. Our previous work was reported for the simultaneous determination of AB and BH using UPLC [53]. Few workers reported for the determination of the related substance of AB by HPLC [54] and BH by spectrophotometric methods [55] as individual articles.

So far, to our present knowledge, the simultaneous determination of related substances of AB and BH in solid pharmaceutical dosages form using UPLC is not reported in any journal or pharmacopeia. Hence we focused on developing a rapid, sensitive and cost-effective method using this advanced technique.

However the thorough literature survey revealed that none of the most recognized pharmacopoeias or any journals includes these drugs in combination for the simultaneous determination of related substances of AB and BH and the information regarding the stability of the drugs is not available. So it is felt essential to develop a liquid chromatographic procedure which will serves a reliable, accurate, sensitive, rapid and stability-indicating RP-UPLC method for the simultaneous determination of related substances of AB and BH in AB+BH capsule.

Regulatory agencies recommend the use of stability-indicating methods [56] (SIMs) for the analysis of stable samples [57]. This requires stress studies in order to generate the potential related impurities under stressed conditions, method development, and validation [58]. With the evident of the International Conference on Harmonization (ICH) guidelines [59], requirements for the establishment of SIMs have become more clearly mandated.

Environmental conditions including light, heat, and the susceptibility of the drug product towards hydrolysis or oxidation can play an important role in the production of potential impurities. Stress-testing can help identifying degradation products and provide important information about intrinsic stability of the drug product [60].

Therefore, herein we report the results of stability study of AB and BH with the aim of determining the extent of the influence of different stress conditions on the stability of drug product.

This manuscript describes the development and subsequent validation of a screening method to simultaneously quantify the related substances of AB and BH by ultra-performance liquid chromatography (UPLC).

2. MATERIALS AND METHODS

2.1. Reagents and materials

AB and BH active pharmaceutical ingredient (API) working standard, and related substances of AB were procured from were procured from Zhejiang Huahai pharmaceutical Co. Limited, Xunqiao, Linhai, Zhejiang, China. Test sample (2.5 mg of AB and 10 mg of BH per capsule) were procured from Market.

Chemical names for all components were listed in Table 1 and chemical structures for all components were shown in Figure 1. Potassium dihydrogen orthophosphate, orthophosphoric acid and acetonitrile were obtained from Merck limited, Mumbai, India. High purity deionized water was obtained from Millipore, Milli-Q (Bedford, MA, USA) purification system.

2.2. Instrumentation

UPLC system (Waters Milford, USA)

equipped with inbuilt auto sampler and binary gradient pump with an on-line degasser was used. The column compartment having temperature control and photodiode array detector (PDA) was employed throughout analysis. Chromatographic data was acquired using Empower software.

2.3. Chromatographic Conditions

Acquity UPLC, BEH C18 (100 x 2.1) mm, 1.7 Hm (Waters Milford, USA) column was used as stationary phase maintained at 40 [degrees]C. The mobile phase involve a variable composition of solvent A (1.36 g of potassium dihydrogen phosphate dissolved in 1000 mL of water, adjusted to pH 3.0 with orthophosphoric acid) and solvent B (acetonitrile). The mobile phase was pumped through the column at a flow rate of 0.3 mL min-1 (Table 2).

The optimum wavelength of 240 nm and 217 nm, which represents the wavelength of maximum response for all components, was selected in order to permit simultaneous determination of related impurities of AB and BH in AB + BH capsules. The stressed samples were analyzed using a PDA detector covering the range of 200-400 nm.

2.4. Solution preparation

2.4.1. Solvent mixture

Mix 8 parts of solvent-A and 2 parts of solvent-B.

2.4.2. System suitability solution

Solution containing a mixture of 350 [micro]g [mL.sup.-1] of AB (equivalent to 250 [micro]g m[L.sup.-1] of amlodipine), 1000 [micro]g [mL.sup.-1] of BH working standard and 2 [micro]g m[L.sup.-1] of benazepril impurity-3 was prepared in solvent mixture.

2.4.3. Standard solution

Solution containing a mixture of 3.5 ng m[L.sup.-1] of AB (equivalent to 2.5 [micro]g m[L.sup.-1] of amlodipine) and 10 [micro]g m[L.sup.-1] of BH working standard was prepared in solvent mixture.

2.4.3.1. Sample solution

Solution containing a mixture of 350 [micro]g m[L.sup.-1] of AB (equivalent to 250 [micro]g m[L.sup.-1] of amlodipine) and 1000 [micro]g m[L.sup.-1] of BH working standard was prepared in solvent mixture.

2.4.3.2. Forced degradation sample solution for specificity study

Multiple-stressed samples were prepared as indicated below. They were chromatographed along with a non-stressed sample solution.

2.4.3.3. Hydrolytic conditions: Acid, base and water-induced degradation

Test solution containing a mixture of 350 [micro]g m[L.sup.-1] of AB (equivalent to 250 [micro]g m[L.sup.-1] of amlodipine) and 1000 [micro]g m[L.sup.-1] of BH were treated with 1N HCl, 1N NaOH and water, respectively. These were subjected to the condition mentioned in Table-4. The solutions were neutralized as needed (1N NaOH or 1N HCl).

2.4.3.4. Oxidative condition: Hydrogen peroxideinduced degradation

Test solution containing a mixture of 350 [micro]g m[L.sup.-1] of AB (equivalent to 250 [micro]g m[L.sup.-1] of amlodipine) and 1000 [micro]g m[L.sup.-1] of BH were treated with 30% w/v [H.sub.2][O.sub.2] in dark under the condition shown in Table 4.

2.5.3.5. Dry heat degradation study

The powdered sample was spread in a flat-bottomed plate to give a homogeneous layer (< 5 mm thick) and subjected to oven under the conditions indicated in Table-4.

2.4.3.6. Photolytic degradation study, Exposed to artificial light

As per guidelines for photo stability testing of new drug substances and products, samples should be exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 watt hours (square meter)-1 to allow direct comparisons to be made between the drug substance and drug product [61]. The powdered sample was spread in a flat-bottomed quartz vessel to give a homogeneous layer (< 5 mm thick) and subjected a sun test chamber and exposed to forced irradiation (at 15 cm from the source) in a sun test chamber fitted with xenon lamp. Sun test meets the ID65 spectral criterion with an optical filter system consisting of a coated quartz glass dish, a window glass filter, and an ID-65 filter used in combination. The sun test's filtered xenon light source is a full spectrum light source containing both visible and UV outputs with a UV cut-on of approximately 320 nm and a spectral distribution corresponding to ID65 per ISO 10977. The sample was exposed with 250 watts [m.sup.-2] for about 22 hours which meets the total espouser of 1.2 million lux hours. (Between 320 nm and 800 nm)

2.4.3.7. Relative humidity degradation study

The powdered sample was spread in a flat-bottomed plate to give a homogeneous layer (< 5 mm thick) and subjected to humidity chamber under the conditions indicated in Table-4.

Simultaneously the common placebo (mixture of all excipients without both drugs), placebo with only AB and placebo with only BH were treated same way as sample treated above in each conditions separately. All these stressed sample and placebo treated under all above stressed conditions were analyzed periodically by UPLC for the appearance of additional impurities and the impurities were identified on the basis of respective placebos and are calculated against the respective main drug component.

3. RESULTS AND DISCUSSION

3.1. Optimization of chromatographic conditions

The possible impurities of AB and BH are very similar to respective drug substances. It is clear from the molecular structures (Fig. 1) that related compounds of AB and BH are acidic and basic in nature. To obtain a good resolution among the impurities and main drug substances we tested different stationary phases considering;

- The feature of stationary phase (RP-[C.sub.8] and RP-[C.sub.18]).

- The particle size of the column (1.7 [micro]m and 2.1 [micro]m).

Considering that AB, BH and their related compounds are a mixture of acidic and basic in nature, we tested following mobile phases with gradient elution,

- K[H.sub.2]P[O.sub.4].[H.sub.2]O (1.36 g [L.sup.-1]) as a buffer (pH

2.5, 3, 3.5, 6.5) combination with acetonitrile.

- K[H.sub.2]P[O.sub.4].[H.sub.2]O (1.36 g [L.sup.-1]) as a buffer (pH 3) combination with methanol.

- K[H.sub.2]P[O.sub.4].[H.sub.2]O (1.36 g [L.sup.-1]) and octanesulfonic acid sodium salt (1 g [L.sup.-1]) as a buffer (pH 3) combination with acetonitrile.

- (N[H.sub.4])[H.sub.2]P[O.sub.4] (1.15 g [L.sup.-1]) as a buffer (pH 3, 3.5) combination with acetonitrile.

- 0.1% orthophosphoric acid as a buffer combination with acetonitrile.

- 0.1% triethylamine adjusted to pH 3.0 with orthophosphoric acid.

3.2. Selection of stationary phase

It is clear from the molecular structure (Fig. 1) that the compounds have polar and non-polar environment. Hence we started the development activity with [C.sub.8]-stationary phase of various manufacturers using different mobile phase. Most of the compounds are polar in nature hence the peak shape for polar compounds shows more tailing. Due to the more tailing, the poor resolutions among the peaks were found. To improve the resolution among the peaks and peak shape, the [C.sub.18]-stationary phase was used. Now the peak shape and resolution among the peaks were improved with Acquity BEH [C.sub.18], 100 x 2.1 mm, 1.7 nm. However, the stationary phase is not only the parameter which gives better separation among all impurities. Mobile phase, pH, and organic modifiers play a very important role which leads to the best separation.

3.3. Influence of organic modifier

Different organic modifiers were tried (acetonitrile, methanol, and tetrahydrofuran) for the better chromatographic separation. With methanol, no satisfactory separation was achieved while with tetrahydrofuran the baseline drift was high hence the response for impurities was reduced. Acetonitrile was found to be better for sensitivity and separation. Hence acetonitile was selected as an organic phase.

3.3.1. Influence of pH of mobile phase buffer

The molecular structure of all components (Fig. 1) implies that few of the compounds possess carboxylic acid and few possess amine functional groups which can readily be ionized under acidic or basic mobile phases. Under acidic mobile phase the acidic components show the drastic change in retention time. Different mobile phase pH ranging from 2.5 to 6.5 was studied. The influence of pH was studied by running the spike sample with all known components with mobile phase pH 2.5 and 3.5. With pH 2.5 the resolution between benazepril impurity-E and benazepril impurity-4 and between amlodipine and benazepril impurity-B was poor whereas in pH 3.5 these resolution were improved with resolution more than 2.0 but at the same time the resolution between Benazepril and benazepril impurity-3 is poor and amlodipine peak is closely eluting with benazepril impurity-3. Hence the middle pH i.e. pH 3.0 was selected. The best separation was achieved with pH 3.0.

3.3.2. Selection of detection wavelength

The UV spectrum in mobile phase exhibits a relative absorption maximum at 240 nm for benazepril hydrochloride, benazepril impurity-E, benazepril impurity-4, benazepril impurity-C, benazepril impurity-F, benazepril impurity-2, benazepril impurity-B, benazepril impurity-D and benazepril impurity-G while at 217 nm for methyl benzenesulfonate, amlodipine impurity-D benazepril impurity-3 and amlodipine impurity-A. Hence these wavelengths i.e. 240 nm and 217 nm were selected for the better sensitivity of the method.

After an extensive study, the method has been finalized on Acquite BEH [C.sub.18], 100 x 2.1 mm, 1.7[micro]m using variable composition of solvent A: 0.01 M K[H.sub.2]P[O.sub.4] in water, adjusted to pH 3.0 with orthophosphoric acid and solvent B: acetonitrile as mobile phase (Table-1). The mobile phase was pumped through the column at the flow rate of 1.0 mL min-1 and column compartment temperature was kept at 40 [degrees]C. The detector response was found maximum at 217 nm and 240 nm; hence the typical chromatogram was recorded at both wavelengths. The typical UPLC chromatogram of sample spiked with all known impurities scanned at 217 nm and 240 nm represent the satisfactory separation of all components among each other (Fig 3).

3.3.3. Method validation

The optimized RP-UPLC method was validated according to ICH guidelines [62], with respect to specificity, accuracy, precision (repeatability and intermediate precision), linearity, range and robustness. System suitability features were also assessed.

3.3.4. System suitability test

The system suitability test was performed according to USP 30 [63] and BP 2007 [64] indications. The observed RSD values at 1% level of analyte concentration were well within the usually accepted values ([less than or equal to] 2%). Theoretical plates, USP tailing factor ([T.sub.f]), and USP resolution ([R.sub.s]) between amlodipine and benazepril impurity-3 were also determined.

3.3.5. Specificity

The peak purity indices of the analytes in stressed sample solutions determined with PDA detector [65-67] under optimized chromatographic conditions, were found to be better (purity angle < purity threshold) indicating that no additional peaks were co-eluting with the analytes and evidencing the ability of the method to assess unequivocally the analyte of interest in the presence of potential interference. Baseline resolution was achieved for all investigated compounds. The FDA guidelines indicated that well separated peaks, with resolution, [R.sub.s] > 2 between the peak of interest and the closest eluting peak, are reliable for the quantification [68]. All the peaks meet this specification. It was observed that in acid, base, water hydrolysis, and peroxide treatment the benazepril impurity C and methyl benzene sulfonate were formed as a major degradation impurity and few unknown impurities indicating that the drug product is sensitive to acid, base, water hydrolysis, and peroxide. The degradation and the results are tabulated in Table 3.

The stability of drugs in analytical solution was checked by preparing sample solution as per method and injected at regular time intervals in the proposed method at room temperature. We verified the formation of additional peaks and found that no additional peaks were formed till 25 hours indicating that the sample solution is stable for about 25 hours.

3.3.6. Linearity and range

The nominal concentration of test solutions for AB and BH were 0.25 mg m[L.sup.-1] and 1 mg m[L.sup.-1,] respectively. Taking into account that typical impurity tolerance level currently is 0.2% and response function was determined by preparing standard solution of each component at different concentration levels ranging form lower limit of quantification to 120% of impurity tolerance level and that identification of impurities below lower level of quantification is not considered to be necessary unless the potential impurities are expected to be unusually potent to be toxic.

The plots of area under the curve (AUC) of the peak responses of the analytes against their corresponding concentrations, they fit straight lines responding to equations. The y-intercepts were close to zero with their confidence intervals containing the origin. The correlation coefficients (r) exceeded 0.98, the acceptance threshold suggested for linearity of procedures for the determination of impurity content in bulk drug [69]. Furthermore the plot of residuals exhibited random patterns with the residuals passing the normal distribution test (p < 0.05), all of which evidenced that the method is linear in the tested range. The regression statistics are shown in Table 4.

3.3.7. Determination of limit of quantification and detection (LOQ and LOD)

The linearity performed above was used for the determination of limit of quantification and detection. Residual standard deviation (o) method was applied and were predicted the values for LOQ and LOD using following formula (a), (b) and established the precision at these predicted levels. The results are tabulated in Table-4.

LOQ = 10[sigma]/S (a)

LOD = 3.3[sigma]/S (b)

Where [sigma] = Standard deviation of response

S = Slope of the calibration curve

3.3.8. Accuracy

Accuracy was evaluated by the simultaneous determination of the analytes in solution prepared by standard addition method. The experiment was carried out by adding known amount of each related impurities corresponding to three concentration levels of 40%, 100% and 150 % of the specification level in sample solution. The samples were prepared in triplicate at each level. The quantification of added analyte (% weight/weight) was carried out by using an external standard of corresponding main drug prepared at the analytical concentration and scanned at both the wavelengths of 240 nm and 217 nm. The experimental results revealed that approximate 95 - 105% recoveries were obtained for all the investigated related compounds. Therefore, based on the recovery data (Table 5) the estimation of related compounds that are prescribed in this report has been demonstrated to be accurate for intended purpose and is adequate for routine analysis.

3.3.9. Method precision and ruggedness

ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) considers ruggedness as the method reproducibility and intermediate precision. The data obtained from linearity study was used for the evaluation of method ruggedness. The method reproducibility was determined from the % RSD of the recoveries obtained from nine samples prepared in triplicates at low (40%), middle (100%) and high (150%) concentration levels of typical impurity tolerance level of corresponding related known impurities. The intermediate precision was determined from the difference in the average recoveries and the difference in the % RSD of the recoveries among the three analysts. The results for all the tested compounds are listed in Table 5 which reveals that the method has good reproducibility and intermediate precision.

3.3.10. Robustness

In order to demonstrate the robustness of the method, system suitability parameters were verified by making deliberate changes in chromatographic conditions, i.e. change in flow rate by [+ or -] 0.03 mL [min.sup.-1], change in pH of the buffer by [+ or -] 0.2 units, change in column oven temperature by [+ or -] 5 [degrees]C, and change in organic composition of mobile phase by [+ or -] 2% of the absolute. The sample spiked with all known impurities at impurity tolerance level was injected and the resolution among the impurities was monitored. The method was demonstrated to be robust over an acceptable working range of its UPLC operational conditions except the change in pH of buffer.

4. CONCLUSION

A stability study was carried out and an efficient UPLC method for the quantification of related substances of AB and BH in drug product was developed and validated. The results of the stress testing of the drug, undertaken according to the ICH guidelines, revealed that the degradation products were formed in hydrolytic (acid, base and water) and oxidative conditions.

Validation experiments provided proof that the UPLC method is linear in the proposed working range as well as accurate, precise (repeatability and intermediate precision levels) and specific, being able to separate the main drug from its degradation products. The proposed method was also found to be robust with respect to flow rate, column oven temperature and composition of mobile phase. The method is sensitive to pH of mobile phase. Due to these characteristics, the method has stability indicating properties being fit for its intended purpose; it may find application for the routine analysis of the related substances of AB and BH in AB+BH capsule.

5. ACKNOWLEDGMENTS

The authors wish to thanks to principal and management of Maulana Azad College, Dr. Rafiq Zakaria campus for providing excellent research facilities.

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Gajanan B. Kasawar, Mazahar N. Farooqui *

Post Graduate and Research Centre, Dr. Rafiq Zakaria Campus, Maulana Azad College, Rouza Bagh, Pin code:431001, Aurangabad, India.

* Corresponding author. E-mail: mazahar_64@rediffmail.com

Article history: Received: 24 February 2012; revised: 16 July 2014; accepted: 18 July 2014. Available online: 19 September 2013.

Table 1. Chemical names of all related impurities of AB and BH.

Compounds                    Chemical name                    Source *

[A] Amlodipine basylate and its impurities

AB          2-[(2-Aminoethoxy) methyl]-4-(2-                     --
            chlorophenyl) -1,  4-dihydro-6-methyl -
            3, 5-pyridine dicarboxylic  acid-3-
            ethyl 5-methyl ester

Imp A       3-Ethyl 5-methyl (4RS)-4-(2-                         P
            chlorophenyl)-2-[[2-(1, 3-dioxo-1, 3-
            dihydro-2H-isoindol-2-yl) ethoxy]
            Methyl]-6-methyl-1,4-dihydropyridine-3,
            5-dicarboxylate

Imp D       3-Ethyl 5-methyl 2-[(2-aminoethoxy) methyl]-4-       P
            (2-chlorophenyl)-6 methyl pyridine-3,
            5-dicarboxylate.

MBS         Methyl benzene sulfonate                             D

[B] Benazepril hydrochloride and its impurities

BH          2-[(3S)-3-[[(2S)-1-ethoxy-1-oxo-4-                   --
            phenylbutan-2-yl]amino]-2-oxo-4, 5-
            dihydro-3H-1-benzazepin-1-yl]acetic acid

Imp B       (3-(1-Ethoxycarbonyl-3-phenyl-(1R)-                  P
            propyl) amino-2, 3, 4, 5-tetrahydro-2-
            oxo-1H-1-(3S) -benzazepine)-1-acetic
            acid monohydrochloride

Imp C       3-(1-Carboxy-3-phenyl-(1S)-                          D
            propyl)amino-2, 3, 4, 5-tetrahydro-2-
            oxo-H-1-(3S)-benzazepine) -1-acetic acid
            (Benazeprilate)

Imp D       (3-(1-Ethoxycarbonyl-3-cyclohexyl-(1S)-              P
            propyl) amino-2, 3, 4, 5-tetrahydro-2-
            oxo-1H-1-(3S)-benzazepine)-1-acetic acid
            monohydro-chloride (Benazepril
            cyclohexyl)

Imp E       3-amino-2, 3, 4, 5-tetrahydro-2-oxo-1H-              P
            1-(3S)-benzazepine)-1-acetic acid
            monohydrochloride

Imp F       t-Butyl-3-amino-2, 3, 4, 5-tetrahydro-               SM
            2-oxo-1H-1-(3S)-benzazepine)-1-acetic
            acid monohydrochloride

Imp G       3-(1-Ethoxycarbonyl-3phenyl-(1S)-                    P
            propyl) amino-2, 3, 4, 5-tetrahydro-2-
            oxo-1H-1-(3S)-benzazepine) -1-acetic
            acid ethyl ester (Benazepril ester)

Imp 2       3-Bromo-2, 3, 4, 5-tetrahydro-2-oxo-1H-              P
            1-(3S) benzazepine-2-one

Imp 3       Ethyl (2R)-2-hydroxy-4-phenylbutyrate                SM

Imp 4       4-Nitrobenzene sulfonyl chloride                     SM

* P--Process impurity, D--Degradation impurity, SM--Starting material

Table 2. Mobile phase program for gradient elution.

Time (min)     Flow     Solvent A   Solvent B
             (mL/min)      (%)         (%)

0.000          0.30        95           5
2.780          0.30        80          20
6.950          0.30        75          25
11.120         0.30        75          25
22.230         0.30        60          40
30.000         0.30        40          60
35.000         0.30        40          60
37.000         0.30        95           5
40.000         0.30        95           5

Table 3. Hydrolytic, oxidizing thermal and photolytic stress conditions.

                     Conditions            Time    Temperature
                                                   ([degrees]C)

Acidic               1.0 N HCl            20 min        80
Basic                1.0 N NaOH           10 min
Hydrolytic           [H.sub.2]O           60 min        80
Oxidation    30% w/v [H.sub.2][O.sub.2]   20 min        80
Thermal                                    12 h        105
Photolytic     250 watt h [m.sup.-2]       22 h
Humidity       40[degrees]C / 75% RH       12 h

              % Degradation

               AB      BH

Acidic        1.13    20.59
Basic        Stable   6.35
Hydrolytic   Stable   5.36
Oxidation     1.56    4.95
Thermal      Stable   1.34
Photolytic   Stable   1.03
Humidity     Stable   1.15

Table 4. Regression statistics and limit of quantification, detection.

Analyte   Conc           Regression        Multiple R   t-Stat
                         equation
          [micro]g
          [mL.sup.-1]

[A] Amlodipine basylate and its impurities

AB        0.101-3.016    y = 14190x + 25   0.99993      448
MBS *     0.020-0.604    y = 6701x - 7     0.99992      365
Imp D     0.039-0.590    y = 14800x + 8    0.99989      343
Imp A     0.031-0.614    y = 12508x - 11   0.99993      433

[B] Benazepril hydrochloride and its impurities

BH        0.411-12.318   y = 7679x - 61    0.99991      439
Imp E     0.082-2.467    y = 4930x - 3     0.99992      383
Imp 4     0.084-0.2524   y = 3266x - 21    0.99993      367
Imp C     0.081-2.419    y = 4916x + 31    0.99993      430
Imp F     0.078-2.347    y = 5325x - 20    0.99994      425
Imp 2     0.085-2.557    y = 6718x + 10    0.99991      381
Imp 3     0.159-2.380    y = 2630x - 8     0.99984      303
Imp B     0.084-2.524    y = 2959x + 10    0.99998      668
Imp D     0.168-2.524    y = 2640x + 25    0.99978      269
Imp G     0.165-2.476    y = 2090x + 2     0.99999      2437

Analyte   P-value    Lower   Upper   LOQ           LOD

                       Confidence    [micro]g      [micro]g
                        interval     [mL.sup.-1]   [mL.sup.-1]

[A] Amlodipine basylate and its impurities

AB        2.03E-32   14142   14277   0.095         0.031
MBS *     5.94E-22   6633    6715    0.010         0.003
Imp D     1.64E-23   14711   14901   0.032         0.011
Imp A     1.25E-24   12406   12533   0.028         0.009

[B] Benazepril hydrochloride and its impurities

BH        4.51E-34   7621    7695    0.323         0.107
Imp E     6.93E-26   4896    4952    0.084         0.028
Imp 4     7.79E-24   3234    3273    0.071         0.024
Imp C     1.69E-26   4918    4968    0.049         0.016
Imp F     1.93E-26   5281    5335    0.071         0.024
Imp 2     1.05E-27   6684    6760    0.061         0.020
Imp 3     6.21E-23   2603    2641    0.140         0.046
Imp B     1.06E-26   2953    2973    0.050         0.016
Imp D     2.33E-22   2639    2682    0.136         0.045
Imp G     3.32E-30   2090    2094    0.017         0.006

* MBS--Methyl benzene sulfonate, LOQ--limit of
quantification, LOD--limit of detection.

Table 5. Precision and recovery.

Analyte   Average recovery (%)       Diff from A-1(%)

          A-1(d)   A-2(d)   A-3(d)   A-2    A-3

[A] Amlodipine basylate and its impurities

MBS *     98.17    101.49   97.30    3.32   0.87
Imp D     100.68   101.56   98.04    0.88   2.64
Imp A     98.80    100.66   99.97    1.86   1.17

[B] Benazepril hydrochloride and its impurities

Imp E     99.36    97.30    98.32    2.06   1.04
Imp 4     97.70    98.28    98.03    0.58   0.33
Imp C     100.60   101.50   100.02   0.90   0.58
Imp F     97.30    97.62    99.66    0.32   2.36
Imp 2     99.40    98.38    99.28    1.02   0.12
Imp 3     97.11    100.10   99.15    2.99   2.04
Imp B     99.58    101.67   100.95   2.09   1.37
Imp D     98.55    101.76   101.00   3.21   2.45
Imp G     100.88   98.62    99.88    2.26   1.00

Analyte   % RSD of recovery        Diff from A-1(%)

          A-1(d)   A-2(d) A-3(d)   A-2    A-3

[A] Amlodipine basylate and its impurities

MBS *     3.16     1.33   1.73     1.83   0.40
Imp D     1.42     3.47   1.23     2.05   2.24
Imp A     1.64     1.91   1.82     0.27   0.09

[B] Benazepril hydrochloride and its impurities

Imp E     0.87     1.10   2.23     0.23   1.13
Imp 4     2.12     1.69   0.86     0.43   0.83
Imp C     2.84     0.78   0.79     2.06   0.01
Imp F     2.46     1.96   0.94     0.50   1.02
Imp 2     0.26     0.51   1.72     0.25   1.21
Imp 3     3.56     3.28   1.94     0.28   1.34
Imp B     3.05     1.84   2.16     1.21   0.32
Imp D     1.16     1.83   0.96     0.67   0.87
Imp G     2.86     1.59   3.25     1.27   1.66

* MBS--Methyl benzene sulfonate.; (d : n = 6); Diff--Difference; A-
1: Analyst-1; A-2: Analyst-2; A-3: Analyst-3
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Author:Kasawar, Gajanan B.; Farooqui, Mazahar N.
Publication:Orbital: The Electronic Journal of Chemistry
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Date:Jul 1, 2014
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