Int J Pharm Pharm Sci, Vol 8, Issue 1, 101-107Original Article


SIMULTANEOUS QUANTIFICATION OF BUPRENORPHINE HCL AND NALOXONE HCL BY VIERORDT’S METHOD

DHANASHREE A. MUNDHEYa*, NIDHI P. SAPKALb, ANWAR S. DAUDa

aCentre for Advanced Research and Innovation (CARIn), Zim Laboratories Ltd. B-21/22, MIDC Area, Kalmeshwar 441501 Dist. Nagpur (M. S.), India, bGurunanak College of Pharmacy, Nari, Kamgarnagar, Nagpur (M. S.), India
Email: dmundhey1990@gmail.com

 Received: 20 Aug 2015 Revised and Accepted: 18 Nov 2015

ABSTRACT

Objective: Development of a simple, rapid and accurate vierordt’s method or simultaneous equation (SE) method for the simultaneous estimation of Buprenorphine HCl (BU) and Naloxone HCl (NA) in bulk and pharmaceutical dosage form. The proposed method was validated according to the guidelines of the International Conference on Harmonization and the Association of Official Analytical Chemists International.

Methods: The method was based on the measurement of absorbance at two wavelengths 289.0 nm and 283.8 nm, λmax of BU and NA in methanol respectively.

Results: Calibration curves were linear in the concentration range of 40-200 µg ml-1 for BU and 40-260 µg ml-1 for NA. The mean recovery, limit of quantification (LOQ) and limit of detection (LOD) for BU were 98.91 %, 0.481 µg ml-1 and 0.158 µg ml-1 and for NA were 98.85 %, 1.283 µg ml-1 and 0.423 µg ml-1, respectively. The method was precise, with a relative standard deviation of less than 2.0 % for both drugs. For robustness, the factors analyzed did not significantly affect the quantification of BU and NA.

Conclusion: The proposed method can be successfully applied for simultaneous estimation of BU and NA in bulk and pharmaceutical dosage form.

Keywords: Vierordt’s method, Simultaneous equation method, Buprenorphine HCl, Naloxone HCl.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Buprenorphine hydrochloride (BU) is chemically known as (6R, 7R, 14S)-17cyclopropylmethyl-7,8-dihydro-7-[(l S)-1-hydroxy-1,2,2trimethylpropyl]-6-0-methyl-6, 14-ethano-17-normorphine hydro-chloride [1-4]. The molecular formula of BU is C29H41NO4, HCl and the molecular weight is 504.1 (fig.1). It is a potent semi-synthetic opiate analgesic with a potency of 20-40 times higher than that of morphine [5]. As an analgesic, it has been used successfully by intramuscular, intravenous or sublingual routes for the treatment of moderate to severe pain as well as chronic pain [6].

Naloxone hydrochloride (NA) is chemically known as Morphinan-6-one, 4,5-epoxy-3,14-dihydroxy-17-(2-propenyl), hydrochloride [7, 2-4].The empirical formula of NA is C19H21NO4, HCl and the molecular weight is 363.84 (fig. 1). It is a potent opioid antagonist and is a competitive antagonist at mu, delta and kappa opioid receptors [6].

BU, opioid narcotic analgesic available in various dosage forms in the US market. BU has abused potential and creates dependence. Thus it is given in combination with its antagonist NA. A few examples of combinatorial dosage forms of BU and NA currently available in the market in different dosage forms by their trade names are SUBOXONE®and QUDICT®sublingual tablet;SUBOXONE®sublingual film and BUNAVAIL®buccal film.

A detailed survey of literature revealed that very few analytical methods have been reported for the estimation of BU [1, 8, 9] and NA [7, 10, 11] individually and in combined dosage form [12, 13] using High Performance Liquid Chromatography (HPLC), Reversed Phase High Performance Liquid Chromatographic (RP-HPLC) and non-aqueous titration method. A single spectrophotometric method has been reported for estimation of BU in raw material but its estimation depended upon an ion pair formation and extraction process which is time-consuming and interference error may occur [9]. An analytical method has also been described for the estimation of BU and NA in biological fluids [14].

No spectrophotometric analytical method has yet been reported in the official compendia for the simultaneous determination of BU and NA in pharmaceutical dosage forms. The present study attempted to develop a rapid, economical, precise and accurate method for simultaneous determination of BU and NA.

The simultaneous estimation method or vierordt’s method permits simultaneous analysis of both compounds without previous separation and extraction procedures. This alternative is simpler and less expensive than HPLC methods [8, 10-12].

A detailed survey of literature also revealed that scientist has previously used this simultaneous estimation method for estimation of metoprolol succinate and olmesartan medoxomil in tablet dosage form [15]; for estimation of dutasteride and tamsulosin hydrochloride in tablet dosage form by vierordt’s method [16]; for estimation of paracetamol and flupirtine maleate in pure and pharmaceutical dosage form [17].

The objective of the present study was to develop a simple, precise, accurate and rapid SE method for the estimation of BU and NA in pharmaceutical dosage form.

MATERIALS AND METHODS

Apparatus

Shimadzu 1800 UV–Vis double beam spectrophotometer with UV probe software and 1 cm matched quartz cells was used.

Materials

Buprenorphine HCl USP and Naloxone HCl USP were procured from Sun Pharmaceuticals Industries Ltd. Analytical grade methanol (Merk Ltd.) was used throughout these experiments.

Standard solutions

Stock solutions of BU and NA were prepared by dissolving 20.0 mg of BU and NA separately in methanol in 50 ml volumetric flasks, and sonicated for 5 min. Final volumes of both the solutions were adjusted with methanol to get stock solutions with the concentration of 400.0 µg ml-1 of BU and NA.

Working standard solutions

Working standard solutions of BU was prepared by pipetting 5.0 ml of BU standard solution of 400.0 µg ml-1 in a 10 ml volumetric flask & working standard solutions of NA was prepared by pipetting 5.0 ml of NA standard solution of 400.0 µg ml-1 in a 20 ml volumetric flask. Final volumes of both the solutions were adjusted with methanol to get working standard solutions with the concentration of 200.0 µg ml-1 of BU and 100.0 µg ml-1 of NA. The concentration range of 40, 60, 80, 100, 120, 140, 160, 180, 200 µg ml-1 was prepared for BU and 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260 µg ml-1 for NA from standard solution for determination of the linearity range.

Sample solution

The sample solution was prepared using developed BU and NA sublingual tablet. A number of 20 accurately weighed tablets were ground into a fine powder using a glass mortar and pestle. A portion equivalent to 4.0 mg of BU and 4.0 mg of NA was accurately weighed and transferred to a 20 ml volumetric flask and volume was made up with methanol. The resulting solution was then sonicated for 5 min & then the final concentration of 200.0 µg ml-1 of BU and NA was obtained.

Method development

Selection of wavelengths

The working standard solutions of BU and NA were then scanned from 250 to 400 nm with the UV spectrophotometer using methanol as a blank. The UV spectrum of BU [18, 19] and NA [20] was shown in the fig.1; thus the λmax of BU and NA was found to be 289.0 nm and 283.8 nm in methanol, respectively & the overlain spectra of BU & NA is shown in fig. 1.

Buprenorphine

Fig.1: Overlain spectra of buprenorphine HCl and naloxone HCl


Fig.2: UV spectra of the mixture containing buprenorphine HCl and naloxone HCl


Fig.3: Calibration graph for buprenorphine HCl


Fig.4: Calibration graph for naloxone HCl


Validation of the concentration range

The absorbance were measured for BU and NA in the concentration range of 40, 60, 80, 100, 120, 140, 160, 180, 200 µg ml-1 and 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260 µg ml-1 at 289.0 nm and 283.8 nm for both the drugs respectively.

Calibration graphs were plotted for BU at 289.0 nm and NA at 283.8 nm at a concentration range of 40-200 µg ml-1 for BU and 40-260 µg ml-1 for NA, respectively as shown in fig. 3 & 4.

Determination of absorptivity value

The absorptivity of each solution was calculated by using the following formula:

Absorptivity = Absorbance/concentration (g/100 ml)

Theory

Simultaneous equation method OR Vierordt’s method

If the sample contains two absorbing drug (X, Y) each of which absorbs at the λmax of the other, then both the drugs can be quantified by using simultaneous equation method [21-23].

For solving this equation the following information is required:

  1. The absorptivities of pure drug X at λ1 and λ2, ax1 and ax2 respectively.

(b) The absorptivities of pure drug Y at λ1 and λ2, ay1 and ay2 respectively.

(c) The absorbance of diluted sample at λ1 and λ2, A1 and A2 respectively.

Let cx and cy be the concentration of X and Y respectively in the diluted sample. Two equation are constructed based upon the fact that at λ1 and λ2 the absorbance of the mixture is the sum of the individual absorbance of X and Y.

At λ1 A1= ax1b cx+ay1b cy.......... (Eq. 1)

At λ2 A2= ax2 b cx+ay2 b cy................  (Eq. 2)

For measurement in 1 cm cells, b=1.

The concentrations of drugs in sample solutions were determined by using the following formula:

Cx = A2ay1-A1ay2/ax2ay1-ax1ay2 (Eq. 3)

Cy = A1ax2-A2ax1/ax2ay1-ax1ay2 (Eq. 4)

Where λ1 and λ2 are the maximum wavelengths of BU and NA,

Cx and Cy are the concentration of BU and NA,

ax1 and ax2 are absorptivities of BU at 289.0 nm and 283.8 nm,

ay1 and ay2 are absorptivities of NA at 289.0 nm and 283.8 nm, respectively.

Method validation

Validation of this new simultaneous spectrophotometric method was carried out as recommended by the International Conference on Harmonization ICH (ICH Q2A & Q2B) guidelines [24, 25] and the Association of Official Analytical Chemists International (AOAC, 2005) [26] for the parameters of specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness.

Specificity

The specificity of the method was evaluated through analysis of a placebo solution. The mixture of inert components was prepared in the usual concentrations employed in developed BU and NA sublingual tablet.

These solutions were analyzed by the proposed method in order to determine if any of the components of the formulation might affect the determinations of BU and NA.

Linearity

Linearity was determined from the calibration graphs plotted for BU and NA at 289.0 nm and 283.8 nm respectively. All spectrophotometric determinations were performed in triplicate and at room temperature (25±2  ̊C). The linear regression was calculated by the method of least squares, and the calibration curves were evaluated by analysis of variance (ANOVA).

Accuracy

The accuracy was calculated based on the percentage of recovery of the known amounts of BU and NA added to the samples [24-26]. Aliquots of the BU and NA standard solutions in concentrations of 200, 250 and 300 µg ml-1 were transferred to 10 ml volumetric flasks containing 5 ml of the sample solution. The volumes were made up with methanol and the drugs were determined in triplicate, using the proposed method.

Precision

The intra-day precision (repeatability) was evaluated by analyzing sample solutions at single concentrations of BU and NA. The analyses were performed in triplicate on the same day. To estimate the inter-day precision, the sample solutions were freshly prepared at the same concentration level for each drug, and the responses were determined in triplicate. This procedure was performed on three consecutive days. The intra-and inter-day precisions are expressed in terms of relative standard deviation (% RSD).

Limits of detection and quantitation (LOD and LOQ)

LOD and LOQ were calculated based on the standard deviation of the response and the slope of the calibration curve. The standard deviation of y-intercepts of regression lines was used as the standard deviation of responses [24-26]. These values were obtained using the following equations:

LOD = 3.3 σ/S (Eq. 5)

LOQ = 10.0 σ/S (Eq. 6)

Where, σ = the standard deviation of the response

S = the slope of the calibration curve

Robustness

Robustness study was carried out by changing the wavelength in ±1 nm of 289.0 nm and 283.8 nm.

RESULTS AND DISCUSSION

Method development

BU and NA are highly soluble in methanol i.e. 42.0 mg/ml and 50.0 mg/ml respectively. Hence it is used as a solvent for standard and sample preparation [27, 28]. The use of vierordt’s or SE method allowed these drugs to be determined simultaneously. The absorbance values were taken at 289.0 nm for BU and at 283.8 nm for NA respectively and then the calibration curves were plotted at their respective wavelength for both the drugs as presented in the fig. 3 & 4 and their absorptivity values were calculated for each concentration as shown in table 1 & 2.

Method validation

Specificity

The specificity test demonstrated that the excipients did not affect the drug determination as depicted in the fig. 5 & 6, respectively. Thus, no interference was observed at 289.0 nm and 283.9 nm indicated that the method is specific.

Linearity

The statistical results of the linear regression for BU and NA are shown in table 3. The coefficient of determination indicated good linearity: 0.997 and 0.998 for BU and NA, respectively. The linearity ranges were 40-200 µg ml-1 for BU and 40-260 µg ml-1 for NA. The absorbance values for these concentration ranges remained between 0.1 and 0.9, conforming to the recommendations of Vogel [23]. The RP–HPLC method for the estimation of BU & NA in pharmaceutical dosage forms shows a smaller range of concentrations [22].

Accuracy

The recovery percentages were 98.91 % and 98.85 % along with % RSD 0.639 and 0.896 for BU and NA, respectively (table 4). These results indicate the accuracy of the method.

Precision

The precision parameters (% RSD) expressed as repeatability (intraday) and as intermediate precision (inter-day) are presented in table 5. For BU and NA, all % RSD were valued were lower than 1% for repeatability in analyses performed during 3 consecutive days, and also for intermediate precision. Thus, the proposed method has good precision in the simultaneous determination of BU and NA.

Table 1: Absorptivity value for BU

Concentration

(µg ml-1)

Absorbance

Absorptivity

Absorbance

Absorptivity

λ1 (289 nm)

λ1(289 nm)

λ2 (283.8 nm)

λ2 (283.8 nm)

40

0.155

38.75

0.143

35.75

60

0.239

39.83

0.219

36.50

80

0.328

41.00

0.302

37.75

100

0.418

41.80

0.384

38.37

120

0.471

39.25

0.432

36.00

140

0.555

39.64

0.508

36.33

160

0.643

40.21

0.590

36.85

180

0.732

40.65

0.669

37.18

200

0.834

41.72

0.769

38.45
 

Mean Absorptivity at 289 nm (λ1)

(M±SD)

40.316±1.06

Mean Absorptivity at 283.8 nm (λ2)

(M±SD)

37.021±0.99

 λ1-maximum wavelength of BU, λ2-maximum wavelength of NA, M-mean and SD-Standard deviation


Table 2: Absorptivity value for NA

Concentration

(µg ml-1)

Absorbance

Absorptivity

Absorbance

Absorptivity

λ2 (283.8 nm)

λ2 (283.8 nm)

λ1 (289 nm)

λ1(289 nm)

40

0.161

40.25

0.142

35.50

60

0.244

40.67

0.217

36.17

80

0.330

41.21

0.294

36.79

100

0.412

41.20

0.369

36.90

120

0.452

37.67

0.404

33.69

140

0.530

37.81

0.473

33.79

160

0.605

37.83

0.542

33.85

180

0.678

37.65

0.607

33.70

200

0.758

37.87

0.678

33.90

220

0.837

38.03

0.749

34.05

240

0.916

38.14

0.820

34.18

260

0.990

38.05

0.887

34.12
 

Mean Absorptivity at 283.8 nm (λ2)

(M±SD)

38.864±1.47

Mean Absorptivity at 289 nm (λ1)

(M±SD)

34.719±1.25

 λ1-maximum wavelength of BU, λ2-maximum wavelength of NA, M-mean and SD-Standard deviation


Fig.5: Graphical evidence that no interference exists between the Buprenorphine HCl and excipients of developed sublingual tablet


Table 3: Statistical and validation parameter

Statistical parameters

BU

NA

λmax

289.0 nm

283.8 nm

Linearity range (µg ml-1)

40-200

40-260

Regression equation (y = mx+c)

Slope (m)

Intercept (c)

Y=0.004x-0.010

0.004

0.010

Y=0.003x+0.020

0.003

0.020

Coefficient of determination (R2)

0.997

0.998

Significant slope (p-valuea)

<0.0001

<0.0001

LOD (µg ml-1)

0.158

0.423

LOQ (µg ml-1)

0.481

1.283

 λmax–maximum wavelength, a Theoretical value of p is based on one–way ANOVA test, at α = 0.05 level of significance


Fig.6: Graphical evidence that no interference exists between the Naloxone HCl and excipients of developed sublingual tablet

Table 4: Recovery data for standard solutions added to the samples analysed by simultaneous equation method

Drug

Theoretical concentration (µg ml-1)

Experimental concentration founda (µg ml-1)

% RSD

Recovery (%)

Mean recovery (%)

% RSD

BU

120

119.69

1.092

99.74

98.91

0.639

125

122.81

0.648

98.25

130

128.35

0.524

98.73

NA

120

119.27

1.151

99.39

98.85

0.896

125

122.56

0.619

98.04

130

129.37

0.666

98.52

RSD–relative standard deviation amean of 3 determinations


Table 5: Intraday and interday precision results

Parameters

Sampling time

BU

NA

Amount present (mg)

Amount present (%)

Mean Amount present (%)

SD

RSD

(%)

Amount present (mg)

Amount present (%)

Mean Amount present (%)

SD

RSD (%)

Intraday precision

0 hr

0.200

100.291

99.42

0.072

0.072

0.265

102.136

102.31

0.074

0.072

4 hr

0.197

98.708

0.266

102.393

8 hr

0.199

99.916

0.265

102.264

Inter day precision

Ist day

0.199

99.421

100.01

1.762

1.761

0.266

102.256

102.77

0.092

0.089

IInd day

0.200

100.083

0.266

102.649

IIIrd day

0.195

97.916

0.267

103.034

SD–standard deviation and RSD–relative standard deviation


Limits of detection and quantitation (LOD and LOQ)

The LODs were 0.158 µg ml-1 and 0.423 µg ml-1 for BU and NA, and the LOQs were 0.481 µg ml-1 and 1.283 µg ml-1 for BU and NA, respectively. These values show that the proposed method has good sensitivity and results are presented in table 3.

Robustness

The responses of BU and NA did not change significantly when the analytical conditions were modified (table 6). These observations confirm the robustness of the method for determination of BU and NA in pure and pharmaceutical dosage form.

Table 6: Results obtained by changing the wavelength±1 nm

Wavelength (nm)

BU

Wavelength (nm)

NA

Amount present (mg)

Amount present (%)

RSD (%)

Amount present (mg)

Amount present (%)

RSD (%)

288

0.196

98.33

0.086

282.8

0.264

101.62

0.072

290

0.196

98.08

0.087

284.8

0.264

101.88

0.072

RSD–relative standard deviation

The objective of this study was to develop and validate a simple & specific UV spectrophotometric method for simultaneous determination of BU and NA. As the λmax of BU and NA are closer together and not even exhibiting a difference of ±10 nm, henceforth no isoabsorption point (λiso) was exhibited as seen in fig. 2, hence estimation of both BU & NA was not possible using absorptive ratio method. Henceforth simultaneous estimation or vierordt’s method was selected for determination of BU and NA in combined dosage forms. This method exhibited precise, accurate and cost effective assay for these drugs in the mixture. Based on previous studies, Mostafavi A et al., (2009) developed and validated a simple isocratic RP-HPLC method for the simultaneous determination of BU, NA dehydrate and its major impurity noroxymorphone in pharmaceutical tablets [12]. The developed method was linear in the range of 0.22–220 µg mL-1 and 0.1–100 µg mL-1 for BU and NA, respectively & the recovery studies for all these three compounds were above 96%. Damodar K et al., (2011) developed and validated a simple & accurate RP-HPLC method for the estimation of BU and NA in pharmaceutical dosage forms.

The developed method was linear in a range of 2.0-12.0 µg mL-1 for both the drugs & the RSD values for accuracy & precision studies were less than 2 % [13]. Sun W et al., (2014) developed a method for the simultaneous determination of NA, BU and norbuprenorphine in human plasma using hollow fiber liquid phase microextraction combined with ultra HPLC & tandem mass spectrometry. The method was linear in the range of 0.1–25 ng mL-1 & the recoveries were in the range of 92.1–106.0% with RSD values were less than 15% [14]. Literature survey revealed a single spectrophotometric method for the determination of BU in raw material or in pharmaceutical formulations [9]. However, this method involved an ion pair complex formation between BU & bromocresol green which resulted into a tedious & more time-consuming process. Though above-mentioned processes for the simultaneous analysis of BU and NA are more sensitive than present method, however all the previous methodologies involved the use of highly sensitive apparatus like HPLC & hyphenated instruments such as UHPLC-MS/MS along with costly organic solvents required for mobile phase development as compared to UV spectrophotometer as employed in this study. The present method using UV spectrophotometer is more cost efficient & less time consuming.

CONCLUSION

A UV spectrophotometric method was developed and validated for the simultaneous determination of BU and NA in bulk and pharmaceutical dosage formulations. The method showed precision, accuracy, LOD, LOQ and robustness, as evaluated according to ICH guidelines. The proposed method proved to be simpler, less expensive, and faster, because no additional pre-treatment of the samples is required prior to the measuring step, thus accelerating the quality-control process. Thus the UV simultaneous equation method was suitable, useful, and an excellent alternative to HPLC to assess quality in routine analysis of BU and NA in drug products.

PERMISSION

The license was granted to M/s Zim Laboratories Ltd. by food & Drug Administration M. S. for working with Narcotic Drugs & Psychotropic Substances.

ACKNOWLEDGEMENT

The authors are thankful to Centre for Advanced Research & Innovation (CARIn), Zim Laboratories, Kalmeshwar Dist. Nagpur (M. S.), India for providing the instrumentation & facilities and License for working with Narcotic opioid analgesic drugs.

ABBREVIATION

SE-Simultaneous equation method, BU-Buprenorphine HCl, NA-Naloxone HCl, LOQ-Limit of Quantification, LOD-Limit of Detection, HPLC-High Performance Liquid Chromatography, RP-HPLC-Reversed Phase High-Performance Liquid Chromatographic, UV-Ultra Violet

CONFLICT OF INTERESTS

All authors have none to declare

REFERENCES

  1. Buprenorphine Hydrochloride. United States Pharmacopoeia 30National Formulary 25 The Official Compendia of Standards United States Pharmacopeial Convention. 2007.
  2. Drugbank Open Data Drug and Drug Target Database. Version 4.1. Available from: http://www.drugbank.ca/drugs/DB01183.  [Article in Press]
  3. Sweetman SC. Martindale the complete drug reference. 36th ed. London: Pharmaceutical Press; 2009.
  4. Ash JE, Budavari S, O’Neill M, Smith A, Heckelman PE, Kinneary J. The Merck Index. 12th ed. USA: Chapman and Hall; 1996.
  5. Heel RC, Brogden RN, TM Speight. Buprenorphine: a review of its pharmacological properties and therapeutic efficacy. Drugs 1979;17:81-110.
  6. Hoskin PJ, Hanks GW. Opioid agonist-antagonist drugs in acute and chronic pain states. Drugs 1991;41:326-44.
  7. Naloxone Hydrochloride. United States Pharmacopoeia 30–National Formulary 25. The Official Compendia of Standards. United States Pharmacopeial Convention; 2007.
  8. Liao CL, Lee CY, Chiu TH. Development and validation of a stability indicating HPLC method for the determination of Buprenorphine in a transdermal patch. J Food Drug Anal 2008;16:1-7.
  9. Amanlou M, Khosravian P, Souri E. Determination of Buprenorphine in raw material and pharmaceutical product using ion pair formation. Bull Korean Chem Soc 2007;28:183-7.
  10. Yaqin LIU, Yonqlianq GAO. Determination of Naloxone hydrochloride nasal sprays and its related substances by HPLC. Pharm J Chin People's Liberation Army 2004;3:181–3.
  11. Panchagnula R, Sharma P, Khandavilli S, Varma MVS. RP-HPLC method and its validation for the determination of naloxone from a novel transdermal formulation. II Farmaco 2004;59:839–42.
  12. Mostafavi A, Abedi G, Jamshidi A. Development and validation of a HPLC method for the determination of Buprenorphine hydrochloride, Naloxone hydrochloride and Noroxymorphone in a tablet formulation. Talanta 2009;77:1415–9.
  13. Damodar K, Bhogineni S, Ramanjaneyulu B. Analytical method development and validation for the simultaneous estimation of buprenorphine HCl & naloxone HCl in pharmaceutical dosage forms by RP-HPLC. Int J Pharm Biol Sci Arch 2011;2:1751–6.
  14. Sun W, Qu S, Du Z. Hollow fiber liquid phase microextraction combined with ultra high-performance liquid chromatography-tandem mass spectrometry for the simultaneous determination of naloxone, buprenorphine and norbuprenorphine in human plasma. J Chromatogr B 2014;951-952:157-63.
  15. Vora BN, Parmar RR, Nayak PP. Development and validation of the simultaneous UV spectrophotometric method for estimation of metoprolol succinate and olmesartan medoxomil in the tablet dosage form. Pharm Methods 2012;3:44-7.
  16. Giriraj P, Sivakkumar T. Simultaneous estimation of durasteride and tamsulosin hydrochloride in tablet dosage form by vierordt’s method. Arabian J Chem 2013;67:1-6.
  17. Giriraj P, Sivakkumar T. New simple spectrophotometric method for the simultaneous estimation of paracetamol and flupirtine maleate in pure and pharmaceutical dosage form. Int J Spectrosc 2014;1-6. doi.org/10.1155/2014/968420. [Article in Press]
  18. Ultraviolet-visible reference spectra. Buprenorphine hydrochloride. Available from: http://www.pmda.go.jp/ english/pharmacopoeia/pdf/sixteenth_edition/JP. [Last accessed on 10 Jul 2015].
  19. SIGMA Product Information. Buprenorphine hydrochloride. Available from: https://www.sigmaaldrich.com. [Last accessed on 10 Jul 2015].
  20. Ultraviolet-visible reference spectra. Naloxone hydrochloride. Available from: http://www.pmda.go.jp/english/ pharma-copoeia/pdf/sixteenth_edition/JP. [Last accessed on 10 Jul 2015].
  21. Beckett AH, Stenlake JB. Practical pharmaceutical chemistry. 4th ed. India (New Delhi): CBS Publishers and Distributers; 1988.
  22. Jain A. Practical in modern pharmaceutical instrumental analysis. 1st ed. India (Pune) Nirali prakashan; 2011.
  23. Jeffery GH, Bassett J, Mendham J. Vogel’s Textbook of Quantitative Chemical Analysis. 5th ed. New York: John Wiley and Sons Publishers; 1989.
  24. Text on validation of analytical procedures: definitions and terminology (Q2A), Triplicate International Conference on Harmonization (ICH) text, ICH Tech coordination; 1994.
  25. Text on validation of analytical procedures: definitions and terminology (Q2B), Triplicate International Conference on Harmonization (ICH) text, ICH Tech coordination; 1995.
  26. AOAC. Association of Official Analytical Chemists. Official Methods of Analysis. 18th ed. Washington DC; 2005.
  27. Elephant Care International. The elephant formulary: Buprenorphine hydrochloride. Available from: http://www.elephantcare.org/Drugs/buprenor.htm. [Last accessed on 10 Jul 2015].
  28. Santa Cruz biotechnology. Product search: Naloxone Hydrochloride. Available from: http://www.scbt.com/ datasheet-203153-naloxone-hydrochloride.Html. [Last accessed on 10 Jul 2015].