Int J Pharm Pharm Sci, Vol 7, Issue 7, 384-389Original Article


COMPARISON OF FT-NIR TRANSMISSION AND HPLC FOR GREEN APPROACH TO DETERMINE PARACETAMOL AND ITS DEGRADATION PRODUCT 4-AMINOPHENOL IN PARACETAMOL TABLETS

AHMED BADR ELDIN*, OMNIA AHMED ISMAIEL**, WAFFA HASSAN**, ABDALLA SHALABY**

*Sigma Pharmaceutical Industries, Quesna, Egypt, **Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University, Egypt
Email: a.badr@sigma-pharm.com

Received: 04 May 2015 Revised and Accepted: 01 Jun 2015

ABSTRACT

Objective: Development and validation of Near infrared (NIR) spectroscopic method for determination of paracetamol and its major degradation product 4-aminophenol in paracetamol tablets and show the agreement between the NIR as a greener technique and the conventional high performance liquid chromatography (HPLC) method, official in British pharmacopeia (BP).

Methods: Calibration model for paracetamol and its degradation product 4-aminophenol was built by utilizing chemometric processing which is the most critical step in the development of specific and robust NIR models. It is based mainly on a partial least square regression fit on the transmission mode using paracetamol, 4-aminophenol and excipient materials of the drug products. The results obtained by NIR spectroscopy were compared with the compendial HPLC method in the BP.

Results: The chosen models had a root mean square error of the cross validation (RMSECV) values of 1.38, 1.42 and coefficient of correlation (r2) of 99.1, 99.05 for paracetamol and 4-aminophenol respectively, which indicates good fitness and accuracy of the model.

Conclusion: The present study showed that NIR could be used with high accuracy for determination of for parent drug and its major degradation product in paracetamol tablets. This proposed technique realizes many of green analytical aspects in developing eco-friendly analytical methods and may replace safely the conventional chromatographic technique without compromising efficacy.

Keywords: Paracetamol, 4-Amino phenol, FT-NIR transmission, GAC (green analytical chemistry), HPLC assay, Validation, PLS model.

INTRODUCTION

The trend of sustainable development requires chemistry to be “clean” or “green.” In the 1990s, therefore, the concept of “Green Chemistry” was proposed, together with the “Twelve Principles of Green Chemistry.”Presently, spectroscopic methods dominate the area of green analytical chemistry [1]. The pursuit in the field of green chemistry is growing dramatically and is becoming a grand challenge for chemists to develop new products, processes and services that achieve the necessary social, economic and environmental objectives due to an increased cognizance of environmental safety, checking environmental pollution, sustainable industrial ecology and cleaner production technologies worldwide [1]. The NIR region spans the wavelength range 12,500–4000 cm-1. In this region, absorption bands correspond mainly to overtones and combinations of fundamental vibrations [2]. In the pharmaceutical sector, several qualitative and quantitative applications of NIR spectroscopy have been described during the manufacturing steps. At the beginning of the manufacturing process, NIR can be used for the identification of active substances and excipients [3–5].

Paracetamol is an old molecules, it is a synthetic non-opiate derivative of 4-aminophenol. Several methods for determination of the drug and its main degradation product have been reported such as spectrophotometric in dosage form [6, 7], fluorimetric in raw material and dosage forms [8], voltammetric in dosage forms and biological fluids [9], high-performance liquid chromatography [10–12], chemiluminescence [13, 14] and capillary electrophoresis [15] methods. However, using NIR for determination of paracetamol has been reported, but using this technique for simultaneous determination of the drug and its parent molecule is a quite new approach.

The aim of this study is to show the agreement between the NIR as a greener technique and the conventional HPLC–UV detection method, official in British pharmacopeia.

MATERIALS AND METHODS

Materials

All materials were supplied by SIGMA pharmaceuticals Corp., Egypt. The commercial samples of paracetamol 500 mg tablets were used and a placebo contains the same raw materials used in the production process, including microcrystalline cellulose, starch, sodium starch glycolate, polyvinylpyrrolidone, magnesium stearate and purified talc. The placebo was used to make serials of dilutions for establishing the calibration model. All materials are of pharmaceutical grade and all chemicals and reagents have been used in the HPLC method are analytical reagent of HPLC grade.

NIR spectroscopy

FT-NIR Spectrometer, MPA Flexible from Bruker Optics (Germany) was used. Pistol grip model with external trigger and LED status lights. It is equipped with two fiber optic probes, NIR probe for liquids "quartz" and NIR probe for solid. Includes 2 m fiber optic cable with Bruker quick connect. Fixed optical path length of 1 mm. NIR probe for solids, probe head length 80 mm, mounting of an integrating sphere for analysis of solid samples in diffuse reflectance and a measurement unit for analyzing highly scattering solid media in transmission. The spectrometer is equipped with a fast, PC-based data system with OPUS/IR FT-IR spectroscopy software package (version 5.0), which was provided by Bruker Optics. OPUS IDENT is a software package designed to identify substances by their NIR spectra while OPUS Quant is designed for the quantitative analysis. For this purpose, OPUS QUANT was used with a partial least square (PLS) fit method. In PLS, the calibration involves correlating the data in the spectral matrix X with the data in the concentration matrix Y. This means that the factoring of the spectral data is more suited for concentration prediction.

Constructing the PLS model

In a first step a PLS regression model was built using calibration samples. The obtained model was chemo metrically validated by leave-one-out cross validation. The final PLS model was described by a selected spectral region, spectra pretreatment and a number of PLS factors. To build the model, 87 different concentrations of tablet preparations were prepared ranged from 50 % to 100% of the labeled amount of the analyte of interest (paracetamol) and from 0.01% to 50 % of the degradation product (4-amino phenol) as demonstrated in table1 where Full cross-validation statistics obtained with calibration models for paracetamol and 4-aminophenol by using of 87 synthetic mixtures of the drug, degradation product and placebo. Each spectrum was the average of 32 scans and the spectrophotometer was operated at a resolution of 8 cm-1.

Spectral data pretreatments

Calibration models were developed using full cross validation. The baseline can drift and maximum absorbance may change. Spectral pretreatments correct these interferences [16, 17]. In this study, reducing of baseline drift and enhancing spectral information has been achieved through chemometric processing include first derivative, second derivative, vector normalization, straight line subtraction and constant offset elimination. The NIR spectra were saved. Measurements were performed by the NIR fiber optic probe for solids. Before measurement, a measurement of the background must be taken and the detector signal shall be checked. In developing method, the measurement conditions shall be determined and saved to be recalled in each measurement time to avoid result variation and ensure high accuracy of the developed analytical procedure. Samples were measured into samples cavities of the equipment tray with keeping minimum illumination in the measurement place by using sample covers to ensure that there was no stray light during measurement. The measurement time for each sample was about 10 seconds per scan and the instrument was operated at a resolution of 8 cm-1.

Reference method

According to the British pharmacopeia, the official HPLC method utilizing a stainless steel column (25 cm × 4.6 mm) packed with octylsilyl silica gel (5 µm) separation column at 35 °C and mobile phase composed mixture of 250 volumes of methanol containing 1.15 g of a 40% (w/v) solution of tetrabutylammonium hydroxide with 375 volumes of 0.05M disodium hydrogen orthophosphate and 375 volumes of 0.05M sodium dihydrogen orthophosphate. The flow rate to be adjusted at 1.5 ml/min and the detection wavelength was set at 254 nm [18].

RESULTS AND DISCUSSION

Method validation

Fig. 1 shows the raw spectra obtained with the calibration samples.

From these spectra, the Quant program selected five regions, automatically; it was between 12000 and 4000 cm-1. All samples have been analyzed by the HPLC official method; Fig.2 shows the resulting chromatogram. As the HPLC reference method is official in the British pharmacopeia, the researcher has performed a verification study include the items of linearity, sensitivity, accuracy and precision for the compendial method while full cross validation has been performed for the proposed NIR method and the validation statistics obtained from the calibration models are recorded in table 1.

Fig. 1: The FT-NIR spectra for paracetamol and 4-aminophenol at different concentrations in synthetic mixtures with placebo


Fig. 2: The resulting chromatogram for paracetamol and its major degradation product (4-aminophenol) under typical chromatographic conditions described in BP


Table 1: Full cross-validation statistics obtained with calibration models for paracetamol and 4-aminophenol by using 87 synthetic mixtures of the drug, degradation product and placebo

Exp. No. 4-Amino phenol Paracetamol
True Prediction
1. 0 0.509
2. 0 -0.2537
3. 0.6 -0.6348
4. 0.8 -0.3979
5. 0.9 1.401
6. 1 0.8788
7. 1.5 0.8175
8. 2 1.622
9. 2.5 2.963
10. 3 3.596
11. 1 -0.4804
12. 0.1 -0.05473
13. 0.1 0.1293
14. 0.1 -0.3529
15. 0.2 -0.01942
16. 0.2 -0.3188
17. 0.2 0.5424
18. 0.3 0.6514
19. 0.3 1.128
20. 0.3 -0.5046
21. 0.4 0.4264
22. 0.4 0.4618
23. 0.4 2.472
24. 0.5 1.471
25. 0.8 1.752
26. 0.8 3.978
27. 0.8 -0.3367
28. 1.1 -0.1098
29. 1.1 3.948
30. 1.1 1.996
31. 1.4 0.1965
32. 1.4 4.379
33. 1.4 2.643
34. 1.8 2.475
35. 1.8 1.734
36. 2.1 0.1965
37. 2.1 1.67
38. 2.3 0.0232
39. 2.3 2.47
40. 2.5 2.458
41. 2.7 1.2
42. 2.9 0.6771
43. 2.9 0.4589
44. 2.9 0.8888
45. 3.1 4.337
46. 3.1 2.031
47. 3.6 5.244
48. 3.6 2.857
49. 4.1 2.648
50. 4.1 2.747
51. 5.1 6.148
52. 5.6 4.998
53. 5.6 6.35
54. 7.6 5.796
55. 8.6 7.784
56. 2 3.09
57. 2 4.611
58. 4 5.348
59. 4 5.002
60. 6 4.471
61. 8 9.408
62. 10 10.64
63. 10 8.691
64. 12 9.784
65. 12 11.73
66. 14 14.97
67. 18 19.1
68. 20 22.01
69. 20 21.89
70. 22 22.39
71. 24 23.62
72. 26 25.21
73. 30 29.51
74. 30 31.25
75. 34 35.7
76. 34 34.35
77. 36 34.56
78. 36 34.08
79 38 39.14
80. 38 36.11
81. 40 41.24
82. 42 41.21
83. 44 47.15
84. 44 42.14
85. 48 45.08
86. 50 47.84
87. 48 50.48

a F Value: reducing such value indicates that the spectra are efficiently represented by the PLS vectors bF Prob: indicates the probability that a standard is a spectral outlier.

Linearity of the reference method

The linearity of calibration curves (peak area vs. concentration) were checked over the concentration ranges from 5-100 µg/ml and from 0.5-10 µg/ml for paracetamol and 4-aminophenol respectively in the drug-matrix solutions, each concentration level was injected 3 times (n=3) and the average peak area was calculated. The resulting curve was found to be linear with correlation coefficients of better than most cases; the limits of detection LOD and the limits of quantitation LOQ, were calculated for the calibration curves as three and ten times of the noise levels for LOD and LOQ, respectively[19]. Table 2 lists the linearity parameters of the calibration curves.

Sensitivity and accuracy of the reference method

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value [19]. The accuracy of the method was tested by analyzing different samples at various concentration levels ranged from 30.30 to 70.70 and 7.28 to 3.12 µg/ml for paracetamol and 4-aminophenol respectively added to drug-matrix used in tablet formulation, each concentration level was injected 3 times (n=3) and the average peak area was calculated. The results were expressed as percent recoveries of the. Table 3 shows that the overall percent recoveries was 100.03 % and 100.26% for paracetamol and 4-aminophenol respectively, with relative standard deviation (RSD) lower than 2% in all cases.

Table 2: Linearity of calibration curve for Paracetamol and 4-Aminophenol in drug-matrix preparation. Number of points in the regression line are 6 for each case

item Calibration range (μg/ml) Correlation coefficient Slope Slope 95% confidence interval for the slopea Intercept Slope 95% confidence interval for the intercepta LOQ (μg/ml) LOD (μg/ml)
Paracetamol 5-100 0.9999 0.810 ±0.0334 0.159 ±1.221 0.347 0.104
4-Aminophenol 0.5-10 0.9998 0.774 ±0.0612 1.555 ±2.545 0.325 0.0975

aConfidence intervals of the slope and the intercept = (SD of the slope or intercept x t), the value of t at 3 degree of freedom and 95% confidence level is 1.33.

Table 3: Accuracy of the proposed HPLC method for the determination of paracetamol and 4-Aminophenol in drug matrix solution

Paracetamol 4-Aminophenol
Quantity added in μg/ml Quantity found in μg/ml
30.30 30.41
40.40 41.02
50.50 49.93
60.60 59.95
70.70 71.02
Average
% R. S. D

Precision of the reference method

According to and ICH [19] guidelines, the precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

Analysis repeatability

It was evaluated by carrying out the analysis of the six homogenous solutions of the same test samples. The determinations were carried out one after the other under conditions as similar as possible.

The relative standard deviation was calculated from the results of the obtained observations as shown in table 4.

Intermediate precision

The intermediate precision of the method was checked by determining precision on a different day using the same number of samples and same concentration range as in repeatability. The relative standard deviation was calculated from the results of the obtained observations. In all cases, the RSD was lower than 2 as shown in table 4.

Table 4: Intra-day and Inter-day precision

Exp. No. Repeatability on day 1 Repeatability on day 2 (Intermediate precision)
Paracetamol 4-Aminophenol
1 99.684% 100.269%
2 98.362% 100.784%
3 99.765% 99.235%
4 100.045% 98.036%
5 98.062% 100.478%
6 100.361% 100.785%
Mean 99.380% 99.931%
SD 0.858 0.995
RSD 0.864% 0.996%

Predictability of the proposed NIR method

The coefficient of correlation (r2) and root mean square error of the cross validation (RMSECV) are essential tools to evaluate the predictability of the obtained chemometric model of the proposed NIR method [20]

Where CHPLC is the amount of paracetamol or 4-aminophenol measured by the reference method, CNIR is the amount of paracetamol or 4-aminophenol as measured by the proposed method and n is the number of samples. The chosen model had a RMSECV value of 1.38, 1.42 and coefficient of correlation (r2) of 99.1, 99.05 for paracetamol and 4-aminophenol as illustrated in fig. 3 & 4, which indicates good fitness and accuracy of the model.

Fig. 3: Regression of the calibration samples for NIR proposed method in cross validation (paracetamol)

Fig. 4: Regression of the calibration samples for NIR proposed method in cross validation (4-aminophenol)

Agreement between the two methods for unknown samples

A simple plot of the results given by a method versus those of the other one is a useful mean to evaluate the validity of the proposed NIR method, however, the data points will usually be clustered near the line and it will be difficult to assess between method differences so that a plot of the difference between the methods against their mean is chosen. This plot of data may be more informative. Fig. 5& 6 shows the distribution of the differences against their mean.

Fig. 5: Illustration of distribution of the differences against their mean in cross validation for paracetamol


Fig. 6: Illustration of distribution of the differences against their mean in cross Validation for 4-aminophenol

CONCLUSION

NIR spectroscopy technique has several advantages making it one of the most favorable techniques according to the GAC principles. In this study, it has been used as an alternative technique to HPLC with UV detector for the determination of paracetamol and its major degradation product 4-aminophenol in tablet dosage form. If an efficient calibration model has been established, it could be used easily for rapid and accurate analysis of a large number of samples. It is a non-destructive method, doesn't need to sample pre-treatment or toxic solvents and reagents and thus it fulfills the green analytical chemistry aspects and suitable for on-line, in-line and off-line production control purposes. Therefore, this technique can replace the conventional HPLC techniques in some applications safely for developing more eco-friendly analytical methods, realizing the green chemistry principles in a direct and clear way.

ACKNOWLEDGMENT

The authors thank SIGMA Pharmaceutical Corp.,-Egypt for technical support.

CONFLICT OF INTERESTS

Declared None.

REFERENCES

  1. Haq N, Iqbal M, Alanazi FK, Alsarra IA, Shakeel F. Applying green analytical chemistry for rapid analysis of drugs: adding health to pharmaceutical industry. Arabian J Chem 2012;2(5):47-54.
  2. Anderson C, Drennen J, Ciurczak E. Pharmaceutical applications of near-infrared spectroscopy. Pract Spectrosc Ser 2008;35:585.
  3. Plugge W, van der Vliest C. The use of near infrared spectroscopy in the quality control laboratory of the pharmaceutical industry. J Pharm Biomed Anal 1992;10(10):797-803.
  4. Gerhausser CI, Kovar K-A. Strategies for constructing near-infrared spectral libraries for the identification of drug substances. Appl Spectrosc 1997;51(10):1504-10.
  5. Vredenbregt M, Caspers P, Hoogerbrugge R, Barends D. Choice and validation of a near infrared spectroscopic application for the identity control of starting materials.: Practical experience with the EU draft Note for Guidance on the use of near infrared spectroscopy by the pharmaceutical industry and the data to be forwarded in part II of the dossier for a marketing authorization. Eur J Pharm Biopharm 2003;56(3):489-99.
  6. Yousefinejad S, Hemmateenejad B. Simultaneous spectrophotometric determination of paracetamol and para‐aminophenol in pharmaceutical dosage forms using two novel multivariate standard addition methods based on net analyte signal and rank annihilation factor analysis. Drug Test Anal 2012;4(6):507-14.
  7. Kamyabi MA. Simultaneous spectrophotometric determination of paracetamol and p‐aminophenol by using mean centering of ratio kinetic profiles. J Chin Chem Soc 2009;56(1):142-9.
  8. Vilchez J, Blanc R, Avidad R, Navalón A. Spectrofluorimetric determination of paracetamol in pharmaceuticals and biological fluids. J Pharm Biomed Anal 1995;13(9):1119-25.
  9. Noviandri I, Rakhmana R. Carbon paste electrode modified with carbon nanotubes and poly (3-aminophenol) for voltammetric determination of paracetamol. Int J Electrochem Sci 2012;7(5):4479-87.
  10. Monser L, Darghouth F. Simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column. J Pharm Biomed Anal 2002;27(6):851-60.
  11. Kartal M. LC method for the analysis of paracetamol, caffeine and codeine phosphate in pharmaceutical preparations. J Pharm Biomed Anal 2001;26(5):857-64.
  12. Oliveira E, Watson D, Morton N. A simple microanalytical technique for the determination of paracetamol and its main metabolites in blood spots. J Pharm Biomed Anal 2002;29(5):803-9.
  13. Easwaramoorthy D, Yu Y-C, Huang H-J. Chemiluminescence detection of paracetamol by a luminol-permanganate based reaction. Anal Chim Acta 2001;439(1):95-100.
  14. Koukli II, Calokerinos AC, Hadjiioannou TP. Continuous-flow chemiluminescence determination of Acetaminophen by reduction of cerium (IV). Analyst 1989;114(6):711-4.
  15. Chu Q, Jiang L, Tian X, Ye J. Rapid determination of acetaminophen and p-aminophenol in pharmaceutical formulations using miniaturized capillary electrophoresis with amperometric detection. Anal Chim Acta 2008;606(2):246-51.
  16. Barnes R, Dhanoa M, Lister SJ. Standard normal variate transformation and de-trending of near infrared diffuse reflectance spectra. Appl Spectrosc 1989;43(5):772-7.
  17. Fearn T. A look at some standard pre-treatments for spectra. NIR news 1999;10(3):10-1.
  18. British pharmacopoeia. British pharmacopoeia commission. London: HMSO publication; 2009.
  19. International Conference on Harmonization. Validation of analytical procedure methodology ICH Q2 (R1); 2005.
  20. Jee R, Moffatt A, Trafford A. Quantification of paracetamol in intact tablets using near-infrared transmittance spectroscopy. Analyst 1998;123(11):2303-6.