Int J Curr Pharm Res, Vol 14, Issue 4, 42-50Original Article


A NOVEL METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTIFICATION OF NEFOPAM HYDROCHLORIDE IN PARENTERAL DOSAGE FORM BY RP-HPLC METHOD

CH. NEELIMA, M. HEMASRI, P. SUSMITHA, S. SRILAKSHMI*

School of Pharmaceutical Sciences and Technologies, Jawaharlal Nehru Technological University Kakinada, Kakinada 533003, Andhra Pradesh, India
*Email: srilakshmisari1984@gmail.com

Received: 20 Apr 2022, Revised and Accepted: 12 Jun 2022

ABSTRACT

Objective: Using RP-HPLC, a simple, accurate, and exact method for estimating the Nefopam Hydrochloride parenteral dose form was devised.

Methods: The isocratic mode of the RP-HPLC method used an Inertsil C8 column as the stationary phase and a mobile phase of potassium dihydrogen phosphate with pH 3.0: Acetonitrile (70:30) at a flow rate of 1 ml/min. With UV detection at 220 nm, a flow rate of 1 ml/min was established.

Results: The developed RP-HPLC technique revealed acceptable linearity (R2 = 0.9998) and good assay results in the concentration range of 0.004–0.08 mg/ml (103.3 percent). Further forced degradation investigations using 0.1N Hydrochloric acid (acid degradation), 0.1NNaOH (base degradation), and 3 percent H2O2 (Hydrogen peroxide) were carried out using RP-HPLC, and percent degradation values were determined. In peroxide degradation conditions, the medication was shown to be unstable.

Conclusion: In compliance with ICH requirements, the developed procedures were validated.

Keywords: Nefopam hydrochloride, RP-HPLC, Method development, Forced degradation studies

© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijcpr.2022v14i4.1982 Journal homepage: https://innovareacademics.in/journals/index.php/ijcpr

INTRODUCTION

Pharmaceutical analysis is particularly important in the quality assurance and control of bulk pharmaceuticals and pharmaceutical formulations. Due to the fast growth of the pharmaceutical business and medication production in many regions of the world, there is a greater demand for novel analytical techniques in the pharmaceutical industry. As a result, the most essential part of the analysis has become the development of analytical procedures. Analytical processes are designed and verified for active pharmaceutical ingredients (API), excipients, drug products, degradation products, related substances, residual solvents, and other chemicals. In quality control laboratories, these analytical methods are used to assure pharmaceuticals' identity, purity, safety, efficacy, and performance [1].

Nefopam Hydrochloridehas the empirical formula C17H19NO. HCl and with the IUPAC name 3,4,5,6-Tetrahydro-5-methyl-1-1-phenyl 1H-2, 5-hydrochloride, 5-Methyl-1-phenyl-1, 3, 4, 6-tetrahydro-5H-benz[f]-2,5-oxazocine hydrochloride [2], is a non-opioid analgesic that inhibits the reuptake of serotonin, dopamine, and noradrenaline. Nefopam is a painkiller. It treats moderate pain, for example, after an operation or a serious injury, dental pain, joint pain, muscle pain, or pain from cancer [3]. This drug is therapeutically used for the relief of moderate to severe pain. The structure of Nefopam hydrochloride is shown in fig. 1[4].

Fig. 1: Structure of nefopam hydrochloride

RP-HPLC is a precise and sensitive method for analyzing the quantitative effects of a variety of medicines. A few approaches for innovative method development and validation for the measurement of nefopam hydrochloride in parenteral dose form have been documented in the literature. The goal of this study was to create and validate Nefopam Hydrochloride using RP-HPLC and conduct forced degradation studies [5].

MATERIALS AND METHODS

The tools used to analyze Nefopam Hydrochloride are listed in table 1.

Table 1: List of instruments/apparatus used

S. No. Name Date handling system Make Model
1 Analytical Balance - Shimadzu AY220
2 Digital pH-Meter - Lobotronics LT-11
3 Sonicator - Enerteck -
4 UV-Visible Spectrophotometer UV probe Shimadzu UV-1800
5 High-Performance Liquid Chromatography LC-Solution Shimadzu LC-2010
6 Millipore vacuum filtration setup (0.45 µm) - - -
7 Calibrated electronic balance - Sartorius -
8 Volumetric flask (10, 20,25,50 ml) - Class borosil -
9 Pipettes (1 micro pipette), 2,5,10 ml - Class borosil -
10 Beakers (50, 100 ml) - Class borosil -

Active pharmaceutical ingredient used

The Nefopam Hydrochloride was obtained from Aurobindo Pharma Ltd. The marketed formulation Accupan®, Aurobindo Pharma Ltd. Hyderabad, India was used as a parenteral dosage form.

Chemicals used

The following table 2 shows the list of chemicals used for the analysis of Nefopam.

Methodology

Selection of detection wavelength

20 mg of Nefopam Hydrochloride was dissolved in water. The spectrum was acquired by scanning the solution from 200 to 400 nm. The overlay spectrum was used for the selection of wavelength for Nefopam Hydrochloride. The detecting wavelength was chosen as the isosbestic point.

Table 2: List of chemicals used

S. No. Name Manufacturer
1. Distilled water In-House Production (Sri Padmavathi School Pharmacy)
2. Hydrochloric Acid Merck Pvt. Ltd (AR Grade)
3. Sodium Hydroxide Merck Pvt. Ltd (AR Grade)
4. HPLC grade Acetonitrile Merck Pvt. Ltd (AR Grade)
5. HPLC grade water Merck Pvt. Ltd (AR Grade)
6. Ammonium acetate Merck Pvt. Ltd (AR Grade)
7. Potassium Dihydrogen phosphate Merck Pvt. Ltd (AR Grade)
8. Orthophosphoric acid Merck Pvt. Ltd (AR Grade)
9. Hydrogen peroxide Spectrum chemicals
10. Methanol Merck Pvt. Ltd (AR Grade)
11. Ethanol Spectrum chemicals
12. Acetone Spectrum chemicals

Selection of column

As the drug is polar, initially, nonpolar column Symmetry®C18 (250*4.6 mm)–5 µm is used. It offers good peak symmetry.

Selection of mobile phase

As the drug is freely soluble in water, it has been selected as one of the solvents. Ammonium acetate and potassium dihydrogen phosphate are used as buffers as their pH correlates to the drug pka value. Acetonitrile is employed to generate a strong peak since the medication molecule is polar and basic.

Selection of pH of mobile phase

Nefopam hydrochloride is basic, and a tertiary amine group is present in it. As it is a bulker molecule, acidic range pH is selected from 3-5. When the pH of the drug is increased, its ionization will be increased.

Selection of diluent

Water was used as the diluent because of the drug’s solubility.

Selection of flow rate

The flow rate should not be more than 2 ml/min. The flow rate that results in the shortest retention period, the lowest back pressures and the best separation will be chosen.

Preparation of solutions

Optimization of Chromatographic conditions

Method development for the analysis of Nefopam Hydrochloride was done by changing mobile phase ratios, buffers, flow rate, columns, and run time. The chromatogram is shown in fig. 2.

Table 3: Optimized chromatographic conditions

S. No. Parameters Condition
1 Column Intersil ® C8-3(250*4.6 mm)-5 µm
2 Pump LC-10
3 Pump mode Isocratic
4 Mobile phase Potassium dihydrogen phosphate: Acetonitrile (70:30)
5 Detection wavelength 220 nm
6 Flowrate 1.0 ml/min
7 Standard and sample concentration 0.04 mg/ml
8 Column oven temperature 30 ° C
9 Run time 20 min
10 Injection volume 10 µl

Table 4: Results of nefopam hydrochloride optimised method

Name RT Area TP TF
NEFOPAM 6.853 2058146 7120 1.7

Fig. 2: Chromatograms of the optimized method

Inference

In this trial, theoretical plates and tailing factors are within the limit; hence this trial was optimized.

Method validation by RP-HPLC

Validation was performed as per the ICH Q2B (R2) guidelines [6, 7]. The method was validated for the parameters like system suitability, specificity, linearity, precision (system precision and repeatability), accuracy, the limit of detection and quantification, robustness, and assay. The stability studies like acid degradation, base degradation, and degradation with hydrogen peroxide were carried out as per ICH guidelines [8].

System suitability parameters [9]

It is performed to verify that the analytical system is working properly and can give accurate and precise results. 5 injections of Nefopam Hydrochloride Standard solution (0.04 mg/ml) were injected into HPLC and system suitability parameters like USP theoretical plate count, and tailing factor, were assessed, which was mentioned in fig. 3.

Fig. 3: Chromatogram of system suitability

Table 5: System suitability parameters for nefopam hydrochloride

Inj. No. RT Peak area Theoretical plates USP tailing factor
1 6.853 1906390 8945 1.2
2 6.853 1906175 8990 1.2
3 6.853 1905960 9053 1.2
4 6.825 1904806 9074 1.2
5 6.824 1903697 9096 1.2
Mean 1905405
SD 1134.3735
%RSD 0.06

Acceptance criteria

Discussion

Because the system suitability parameters were within the limitations, the parameters for the optimized method could be used to validate the method.

Specificity

The specificity of the method is performed by separately injecting the blank, placebo, and sample solutions. In the improved approach, the interference is checked. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So, this method was said to be specific.

Discussion

From the above chromatogram, it can be concluded that there is no interference between the blank and placebo in the method.

Linearity

Weigh accurately and transfer about 20.0 mg of Nefopam Hydrochloride working standard into 50 ml volumetric flask, add 20 ml of diluent, sonicate with intermittent shaking to dissolve the contents, and dilute to 50 ml with diluent and mix well. Further, dilute 5 ml of this solution to a 50 ml volumetric flask and dilute to volume with a diluent.

Standard solutions of 50%, 100%, and 150% concentrations were prepared by taking 0.5, 1.0, and 1.5 ml each from two standard stock solutions to make up to 10 ml.

Linearity for the concentration range 0.004-0.08 mg/ml was established by plotting concentrations mentioned in fig. 7.

Discussion

Six linear concentrations of Nefopam (50-300 µg/ml) were injected in a duplicate manner. Average areas were mentioned above, and linearity equations obtained for Nefopam was y = 7189x+28445; the correlation coefficient obtained was 0.999.

Precision

Preparation of solutions

Preparation of Nefopam Hydrochloride Standard stock solution-I (0.4 mg/ml): 20 mg of Nefopam API was weighed and transferred into a 50 ml volumetric flask and dissolved in freshly prepared diluent and made up to the volume.

Preparation of Nefopam Hydrochloride Standard stock solution-II (0.04 mg/ml): 5 ml was pipetted out from stock-I solution into 50 ml volumetric flask and made up to the mark with the diluent.

Preparation of Nefopam Hydrochloride Sample stock solution-I (0.4 mg/ml): 2 ml of Nefopam sample was transferred into a 20 ml volumetric flask and dissolved in freshly prepared diluent and made up to the volume.

Preparation of Nefopam Hydrochloride Sample stock solution-II (0.04 mg/ml): 2 ml was pipetted out from the stock-I solution into a 50 ml volumetric flask and made up to the mark with the diluent.

Method precision: 6 individual sample preparations are injected into the system.

Table 6: Linearity profile by RP-HPLC

Concentration (µg/ml) Peak area of nefopam
0 0
50 488555
100 995266
150 1493280
200 2020341
250 2492454
300 3036166

Table 7: Summary of regression equation by RP-HPLC

Line equation y = 7189x+28445
Correlation coefficient (R2) 0.999
y-intercept (C) 3030.8
Slope (m) 7189

Fig. 4: Chromatogram of blank

Fig. 5: Chromatogram of placebo

Fig. 6: Overlay chromatograms of blank, placebo, and nefopam hydrochloride

Fig. 7: Calibration curve of Nefopam HCl, On X-axis and the corresponding peak area on Y-axis. Statistical parameters like correlation Coefficient (R2), line equation including slope (m), and y-intercept (C) were determined

Table 8: Results for precision by RP-HPLC

Sample name Volume taken (ml) Dilution Peak area Mg/ml % assay (w/w)
NFM-PREP-1 2 50 1972416 10.3 103.2
NFM-PREP-2 2 50 1973948 10.3 103.2
NFM-PREP-3 2 50 1975734 10.3 103.3
NFM-PREP-4 2 50 1974810 10.3 103.3
NFM-PREP-5 2 50 1978904 10.3 103.5
NFM-PREP-6 2 50 1980098 10.4 103.6
Average-103.3, SD-0.154, % RSD-0.1

Discussion

From a single volumetric flask of working standard solution, six injections were given, and the obtained areas were mentioned above. Average area, standard deviation, and % RSD were calculated. % RSD obtained as 0.1 %respectively for Nefopam. As the limit of Precision was less than “2” the system precision was passed in this method.

Accuracy

Standard preparation (0.04 mg/ml)

Transfer about 20.0 mg of Nefopam Hydrochloride working standard to a 50 ml volumetric flask, add 20 ml of diluent, sonicate with intermittent shaking to dissolve the contents, then dilute to 50 ml with diluent and mix it well. Combine 5 ml of this solution with 50 ml of water to make a 50 ml solution. Fill a volumetric flask halfway with diluent and dilute to volume.

Preparation of 50% Spiked solution: In a 10 ml volumetric flask, 0.5 ml of sample stock solution was pipetted out, and 1.0 ml of each standard stock solution was pipetted out and made up to the mark with diluent.

Preparation of 100% Spiked solution: 1.0 ml of sample stock solution was pipetted into a 10 ml volumetric flask, along with 1.0 ml of each standard stock solution, and diluent was added to make up to the mark.

Preparation of 150% Spiked solution: 1.5 ml of sample stock solution was pipetted into a 10 ml volumetric flask, followed by 1.0 ml of standard stock solution pipetted out and diluent prepared up to the mark.

Placebo solution of 0.2 ml was spiked with 50,100.150% level solutions of standard stock solution and analyzed. 10 µl of each of the above solutions were injected into the chromatographic system and peak areas were noted. Calculated the individual recovery and mean recovery values.

Acceptance criteria

Each level’s percent recovery should be between 98.0 and 102.

Table 9: Accuracy results for nefopam hydrochloride

Recovery levels Standard volume taken Dilution Area Amount added (mg/ml) Amount found (mg/ml) % Recovery
50% 2.5 ml 50 1036562 0.021 0.021 103.6
100% 5 ml 50 2024827 0.042 0.042 101.2
150% 7.5 ml 50 3042072 0.063 0.063 102.1
Average-102.1, SD-1.3495, % RSD-1.3

Discussion

Three levels of Accuracy samples were prepared by the standard addition method. Triplicate injections were given for each level of accuracy and mean % Recovery was obtained as 101 % for Nefopam.

Limit of detection and limit of quantification

The LOD and LOQ of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. “LOD and LOQ are related to both the signal and the noise of the system and is usually defined as a peak whose signal-to-noise (S/N) ratio are at least 3:1 for LOD and 10:1 for LOQ” [10]. The results were shown in table 10.

Robustness

Typical variations includea change in flow rate (±0.1 ml of optimized flow rate), change in pH of the buffer (±10%), and column oven temperature (25 °C and 35 °C) was assessed.

Discussion

Robustness conditions like Flow minus (0.9 ml/min), Flow plus (1.1 ml/min), temperature minus (25 °C), and temperature plus (35 °C) were maintained, and mobile phase pH (2.9 and 3.1) samples were injected in a duplicate manner. System suitability parameters were not much affected, and all the parameters were passed. % RSD was within the limit.

Solution stability

Sample and Standard solutions are prepared at the method concentration (0.04 mg/ml) and are injected for 2,4,8,12,15,20,24 h time intervals their peak areas were noted and % variation is calculated.

Table 10: Results for lod and loq of aclidinium bromide and formoterol fumarate

Drug LOD (µg/ml) LOQ(µg/ml)
Nefopam hydrochloride 0.0014 0.0043

Table 11: Summary of robustness data

Parameter Condition System suitability parameters % RSD
Theoretical plates USP tailing factor
Change in flow rate (±0.1 ml/min) 0.9 ml/min 9134 1.2 0.08
1.1 ml/min 8213 1.2 0.04
Change in temperature (25 °C and 35 °C) 25 °C 8844 1.2 0.04
35 °C 8959 1.2 0.06
Change in Buffer pH (2.9,3.1) 2.9 8444 1.2 0.05
3.1 8958 1.2 0.05

Table 12: Sample solution stability

Solution stability time Nefopam hydrochloride
Peak area % Variation
Initial 1982592 NA
2 h 1986145 0.17
4 h 1985929 0.18
8 h 1984987 0.23
12 h 1983594 0.30
15 h 1983315 0.32
20 h 1982592 0.35
24 h (Benchtop) 1982592 0.78

Table 13: Standard solution stability

Solution stability time Nefopam hydrochloride
Peak area % Variation
Initial 1934233 NA
2 h 1922769 0.59
4 h 1920096 0.73
8 h 1919829 0.74
12 h 1919829 0.90
15 h 1914941 1.00
20 h 1914787 1.01
24 h (Benchtop) 1904788 1.53

Assay of formulation by RP-HPLC

Calculated the content of Nefopam Hydrochloride by using the following formula.

Content in mg/ml

=

= 10.3 mg/ml

= 103.2%

Acceptance criteria: 90-100 %. Assay results were satisfactory and found to be within the limits.

Forced degradation studies

Preparation of solution

Preparation of 1N Hydrochloric acid: 0.85 ml of HCL was taken in a 10 ml volumetric flask and made up to the mark with carbon dioxide-free water.

Preparation of 1N Sodium Hydroxide: 0.40g of NaOH was taken in a 10 ml volumetric flask and made up to the mark with carbon dioxide-free water.

Preparation of 3% Hydrogen peroxide: 10 ml was taken in a 100 ml volumetric flask from 30% hydrogen peroxide and made up to the mark with water.

Acid degradation

1 ml of sample solution was pipetted into a 10 ml volumetric flask and added 1 ml of 1N HCl. Then kept in a hot air oven at 60 °C for 3 h. After 3hours the flask is taken out and added 1 ml of 1N NaOH and made up to the mark with the diluent. From the above solution, 2 ml was pipetted out into a 50 ml volumetric flask and made up to the mark with diluent, and then injected into HPLC.

Base degradation

1 ml of sample solution was pipetted into a 10 ml volumetric flask and added 1 ml of 1N NaOH. Then kept in a hot air oven at 60 °C for 3 h. After 3 h the flask is taken out and added 1 ml of 1N HCl and made up to the mark with the diluent. From the above solution, 2 ml was pipetted out into a 50 ml volumetric flask and made up to the mark with diluent, and then injected into HPLC.

Degradation with hydrogen peroxide

1 ml of sample solution was pipetted into a 10 ml volumetric flask and added 1 ml of 3% Hydrogen peroxide, made up to the mark with diluent, and then injected into HPLC.

Fig. 8: Acid degradation (0.1 N HCl)

Fig. 9: Base degradation (0.1 N HCl)

Table 14: Calculation of forced degradation studies

Condition Area % Degradation
Basic condition 1902678 0.19
Acidic condition 1896187 0.53
Hydrogen peroxide 1868844 1.96

Fig. 10: Peroxide degradation (0.1 N HCl), The above fig. are the chromatograms of acid, base, and peroxide degradation studies.

DISCUSSION

Forced degradation studies reveal the % degradation values of Nefopam Hydrochloride, and the drug was found to be unstable at peroxide conditions.

CONCLUSION

Analytical method RP-HPLC was developed and validated to analyze Nefopam Hydrochloride in the parenteral dosage form [11].

RP-HPLC method in isocratic mode involved the utilization of Intersil C8 column as stationary phase and mobile phase constituting potassium dihydrogen phosphate with pH 3.0: Acetonitrile (70:30) at a flow rate of 1 ml/min with UV detection at 220 nm was developed. The developed RP-HPLC method demonstrated good linearity in the concentration range of 0.004-0.08 mg/ml (R2= 0.9998) and good assay results (103.3%). Further forced degradation studies were carried out by RP-HPLC using 0.1N HCL (acid degradation), 0.1N NaOH (Base degradation), and 3%H2O2 (Hydrogen peroxide), and % degradation values are determined. The drug was found to be unstable in peroxide degradation conditions.

All the methods were validated as per the ICH guidelines and can be used for regular analysis of Nefopam Hydrochloride in Quality Control Laboratories.

ACKNOWLEDGMENT

The author wishes to thank AUROBINDO PHARMA Pvt Ltd., Hyderabad, for providing the facility to complete the work.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Analytical chemistry [homepage]. Available from: http://www.collinsdictionary.com /dictionary/english/analytical-chemistry.

  2. DRUGBANK. Available from: https://go.online.drug bankDrugbank.com/salts/DBSALT002173 [Last accessed on 15 Jun 2022]

  3. The NHS website available. https://www.medicines/nefopam.

  4. https://www.sigmaaldrich.com/IN/en/product/sigma/sml0909.

  5. Skoog. West and holler. Fundam Princ Anal Chem Eight Ed; 2013. p. 5.

  6. International Conference on Harmonization on Validation of Analytical Procedures: Text and Methodology; 2005.

  7. Pharmaceutical development. Q8 (R2). ICH harmonized tripartite guideline. Current Step 4 version; Aug 2009.

  8. International Conference on Harmonization. Stability guidelines. Vol. Q1. Stability testing guideline for pharmaceutical development; 2019. doi: 10.22214/ ijraset.2019.12163.

  9. USP. Chromatography system suitability. USA Pharmacopeial Convention; 2009.

  10. Snyder LR, Kirkland JJ, Joseph L. Glajch quantitation (Including trace analysis). 2nd ed. Wiley India Pvt, Ltd. 2012. p. 645-9.

  11. Ahuja S, Jespersen N. editors. Compr Anal Chem. 2006. p. 47.

  12. Chauhan A, Mittu B, Chauhan P. Analytical method development and validation: a concise review. J Anal Bioanalytical Tech 2015;6:1.

  13. Schmauser B. Analytical method development. Training workshop. Pharmaceutical development with a focus on Paediatric Formulations. World Health Organization; June 2010.

  14. Validation of analytical procedures: text and methodology. ICH Q2. ICH harmonized tripartite guideline. Registration of Pharmaceuticals for human use. November 2005;R1:4.

  15. General chapters 1225, validation of compendial methods Convention, Inc. United States Pharmacopeia. Rockville, MD: The United States Pharmacopeial; 2007. p. 30.

  16. Sales UV [handbook]. International Marketing Division. 2006 version. Vol. 2. Chapter 1; 2006. p. 1-2.

  17. Beckett AH, Stenlake JB. Practical pharmaceutical chemistry. 4th ed. Part Two: CBS Publishers and Distributors Pvt Ltd; 2007. p. 157-66, 278-300.

  18. Mohsena Am, Hayam M Lotfyb, Amr M Badaweyb, Hesham Salema, Sonia Z Elkhateeb. Application of three novel spectrophotometric methods manipulating ratio Spectra for resolving a pharmaceutical mixture of chlorphenoxamine Hydrochloride and caffeine. International Journal of Pharmacy and Pharmaceutical Sciences. 2013;Suppl 1:5.

  19. J Karpinska. Basic principles and analytical application of derivative spectrophotometry. Joanna Karpinska Institute of Chemistry; 2012. p. 254-70.

  20. Ahuja S, Rasmussen H. HPLC method development for pharmaceuticals. Elsevier Inc; 2007. p. 1-3.

  21. Kromidas S. Practical problem-solving in HPLC. Wiley-VCH Verlag; 2000.

  22. Ahuja S. Chromatography and separation science. Vol. 4. Academic Press; 2003. p. 188-99.

  23. Synder LR, Kirkland JJ, Glajch JL. Practical HPLC method development. 2nd ed. New York: Wiley Interscience; 2007. p. 1-20.

  24. Kazakevich Y, Lubrutto R. HPLC for pharmaceutical scientists. New York: John Wiley & Sons Inc; 2007. p. 107.

  25. http://www.ich.org/file admin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1 A_R2/Step4/Q1A_R2 Guideline.pdf.

  26. Shinde NG, Bangar BN, Deshmukh SM, Sulake SP, Dipak Sherekar. Pharmaceutical forced degradation studies with regulatory consideration. Asian J Res Pharm Sci. 2013;3(4, October-December):178-88.

  27. Nefopam hydrochloride. Available from: http: www.druginfosys.com/drug aspx?

  28. Evans MS, Lysakowski C, Tramer MR. Nefopam for the prevention of postoperative pain: quantitative systematic review. Br J Anaesth. 2008;101(5):610-7. doi: 10.1093/bja/aen267, PMID 18796441.

  29. Singh S, Sharma N, Singla YP. Development and validation of UV spectrophotometric method for quantitative estimation of nefopam hydrochloride in polymethacrylate nanospheres. Int J Pharm Pharm Sci 2016;8(1):414-9.

  30. Aymard G, Warot D, Demolis P, Laville I, Diquet B. Sensitive determination of nefopam and its metabolite desmethyl-nefopam in human biological fluids by HPLC. J Pharm Biomed Anal. 2002 Nov;30(4):1013-21. doi: 10.1016/s0731-7085(02)00443-0, PMID 12408892.

  31. Hong C, ZHANG Li-long HPLC. Determination of nefopam hydrochloride. Acta Acad Med Jiangxi. 2005;3.

  32. Balandin VV, Gorobets ES. Postoperative analgesia with nefopam and non-steroidal anti-inflammatory drugs in patients after surgery for tumors of head and neck. Anesteziol Reanimatol. 2014;1:40-3. PMID 24749308.

  33. Eremenko AA, Sorokina LS, Pavlov MV. The use of central acting analgesic nefopam in postoperative analgesia in cardiac surgery patients. Anesteziol reanimatol. 2013;2:78-82. PMID 24000658.

  34. Chanques G, Sebbane M, Constantin JM, Ramillon N, Jung B, Cisse M. Analgesic efficacy and haemodynamic effects of nefopam in critically ill patients. Br J Anaesth. 2011;106(3):336-43. doi: 10.1093/bja/aeq375, PMID 21205626.

  35. Richards BL, Whittle SL, van der Heijde DM, Buchbinder R. Efficacy and safety of neuromodulators in inflammatory arthritis: a Cochrane systematic review. J Rheumatol Suppl. 2012;90:28-33. doi: 10.3899/jrheum.120339, PMID 22942326.

  36. KaizhiLu HS, Chen Y, Li M, Tian Guopin, Chen J. Ondansetron does not attenuate the analgesic efficacy of nefopam. Int J Med Sci. 2013-01-01.

  37. Taniguchi Y, Ali SZ, Kimberger O, Zmoos S, Lauber R, Markstaller M. The effects of nefopam on the gain and maximum intensity of shivering in healthy volunteers. Anesth Anal. 2010;111(2):409-14. doi: 10.1213/ ANE.0b013e3181e332bb, PMID 20529984.