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FT-NIR spectroscopy for rapid and simple determination of nimesulide in rabbit plasma for pharmacokinetic analysis
Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 157–162
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
FT-NIR spectroscopy for rapid and simple determination of nimesulide in rabbit plasma for pharmacokinetic analysis P.V. Ajayakumar a,∗ , Debabrata Chanda b , Anirban Pal b , Mahendra P. Singh b , A. Samad c a
Analytical Chemistry Department, CSIR – Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India In-vivo Testing Facility, Molecular Bioprospection Department, CSIR – Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India c Microbiology and Plant pathology Department, CSIR – Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India b
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 19 September 2011 Accepted 19 September 2011 Available online 24 September 2011 Keywords: Pharmacokinetics Bio-availability FT-NIR Nimesulide Rabbit
a b s t r a c t High-throughput analysis of a large number of samples for pharmacokinetic study is necessary in drug development and pharmacovigilance. Usually, drug quantification for pharmacokinetics and bioavailability is achieved through matrix extraction and HPLC analysis, which is time, labour and cost intensive method. A prompt and solvent free method is the quest for such analysis in the present times. Pharmacokinetic analysis of nimesulide from plasma samples of rabbits through Fourier transform near infrared (FT-NIR) spectroscopy analysis combined with partial least squares (PLS) regression model was undertaken with validation through HPLC analysis. Pharmacokinetic parameters obtained through FTNIR and HPLC were found to be statistically similar with errors below the acceptable limits. The study demonstrates the use of FT-NIR for pharmacokinetics and bio-availability studies. This high throughput method analyses more than 50 samples in an hour without solvents usage and provide ample scope for automation and commercial utilization. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Near infrared (NIR) spectroscopy is a noninvasive, relatively low cost optical technique, portable, useful for real-time measurement of changes in tissues in relation to oxygenation and perfusion [1], body fat [2], qualitative and quantitative measurements of various chemicals in foods, pharmaceuticals, materials, medical, agricultural produce etc. [3]. In addition, Fourier transform near infrared (FT-NIR) is extensively used in different fields of the pharmaceutical industry like prediction of tablet properties of raw-mixed powders before compression [4]; quantitative measurement of components in intact tablets [5,6]; raw materials, intermediate and finished product forms including gels [7] and development of solid dosage forms [8] besides others. Recently, FT-NIR has extensively being explored for studies in urology [1,9]; sports medicine [10], realtime study of brain vascular and metabolic activities [11]. Much of the appeal of NIR technique is because of the fact that wealth of chemical and physical information can be obtained with in seconds often without the need for any sample preparation. Although Fourier transform infrared spectroscopy (FT-IR) has been used for determining molecular concentrations of different bio-active analytes in various biological matrices like blood, serum, plasma and urine [12], the use of FT-NIR in the bio-analysis of
pharmacophore in biological samples like blood, urine, plasma or serum samples has not yet been reported. There is a tremendous need and scope for novel and high throughput analytical method for bioanalysis in the course of drug discovery and development [13]. Hence in the present experiment, we have done pharmacokinetics study of nimesulide in rabbit through FT-NIR analysis of plasma samples and validated the same through HPLC analysis. Nimesulide (4-nitro-2-phenoxymethanesulfonanide) was chosen because of its continuing use in India and other developing countries as a prominent, selective cyclooxygenase (COX-2) inhibitor in inflammatory conditions as well as the easiest availability of high-performance liquid chromatography (HPLC) methods and IR data for the purpose of cross validation. The pharmacokinetics aspects of nimesulide have been documented in human [14] and some animal species using traditional methods [15,16], but none has explored the novel method of calculating kinetic parameters from FT-NIR generated data. Rabbits were preferred to other rodents because its heart and cardiac circulation are closely similar to those of human [17].
2. Materials and methods 2.1. Animals
∗ Corresponding author. Tel.: +91 522 2359632; fax: +91 522 2342666. E-mail address: [email protected] (P.V. Ajayakumar). 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.09.022
Adult male and female New Zealand white rabbits (2.5 kg body weight), six in numbers, being maintained at the in vivo testing
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facility, CIMAP, Lucknow, India were used for the study. The animals were acclimatized to the experimental environment for 7 days before the actual experimentation. The animals were re-used after a wash period of 21 days. The protocols used were duly approved by Institutional Animal Ethics Committee (IAEC) through CPCSEA, Government of India. 2.2. Chemicals Nimesulide and Cremophore EL were obtained from Sigma Chemicals, India. HPLC grade solvents were obtained from Merck India Ltd., India 2.3. Treatment of animals with nimesulide and collection of samples
detector was set at 210 nm and the analyses were carried out using the same series of the extracted plasma samples used in the FT-NIR analysis. 2.7. Kinetic analysis Pharmacokinetic parameters were calculated following the procedure as reported earlier [18] using the software PK87. The whole experiments were conducted three times in order to verify the reproducibility of the method. The spectra obtained from first and second rounds of experiments were used for calibration and validation of the PLS model. The spectra from the third round of the experiment were used only for prediction, and the predicted values were compared with the results from the HPLC analysis. 2.8. Calibration in raw plasma samples
A clear aqueous solution of nimesulide was prepared in 10% cremophore EL in 0.9% sodium chloride (NaCl) in water. Nimesulide was injected at 5 mg kg−1 body weight intraperitonially (i.p.) to the rabbits. Blood samples were collected in heparinised tubes, in duplicate, at 0, 5, 15, 30, 60, 120, 240, 360 and 480 min after the administration of the drug. Clear plasma samples were collected from the blood samples after centrifugation at 5000 × g for 5 min at 4 ◦ C.
A stock solution of nimesulide (5 mg ml−1 ) was prepared in double distilled water containing 0.9% NaCl and 12.5% cremophore EL. Two fold serial dilution of this nimesulide stock solution ranging from 2.5 mg ml−1 to 0.02 mg ml−1 was prepared in freshly collected rabbit plasma. Four replicates of the eight dilutions were subjected to FT-NIR analysis using plasma as blank.
2.4. FT-NIR analysis
3. Results and discussion
The plasma samples thus obtained were used directly for the Fourier transform near infrared (FT-NIR) analysis. The FT-NIR absorbance spectra from 10,000 cm−1 to 4000 cm−1 at a resolution of 4.0 cm−1 (using Antaris II Analyzer, Thermo Fisher Scientific, USA) in transmission mode, using indium gallium arsenide (InGaAs) detector were recorded for each sample. To reduce sampling error, the three vials (0.5 mm cell with 170 l rabbit plasma) of samples from each plasma sample prepared in the previous step were analysed individually. The plasma obtained from the blood collected at zero (0) minute (just before the administration of the drug) was considered as the blank and the background measurements were taken using the blank before every spectral measurement. Data acquisition, spectral mathematical treatments and partial least squares (PLS) regression were done using TQ Analyst Software (Thermo Fisher Scientific, USA). The second derivative of absorbance spectra smoothed with Norris derivative (segment length 19, gap between points 2) was used for all regression work. For path length corrections, standard normal variate (SNV) path length was chosen.
The raw absorbance spectra recorded for nimesulide in rabbit plasma, taken at different time intervals after the i.p. injection, are shown in Fig. 1. The FT-NIR data from first and second rounds of the experiment were randomly split into calibration set (64 samples, 4 rabbits) and validation set (32 samples, 2 rabbits). All the data from the third round of the experiment were used as prediction set (48 samples, 6 rabbits). The two validation animals were selected randomly in each time interval. The visual analysis of the second derivative spectra gave peaks and dips with an indication that at wave number ranges 6430–6360 cm−1 (Fig. 2), 5364–5290 cm−1 and 4849–4833 cm−1 the y-axis values change in the same way as the concentration of the drug, quantified through HPLC analysis, in the collected blood samples. At wave numbers ranging between 5364 and 5290 cm−1 , the spectra showed a dip. The FT-IR spectrum of the pure drug showed prominent peaks at wave numbers 3284, 1590, 1488, 1342, 1282 and 1247 cm−1 [19]. So the significant contribution of the FT-NIR band obtained at 6430–6360 cm−1 could be from the combination of overtones arised from N–H stretching and N–H deformation. Similarly, the major contribution for the band at 4849–4833 cm−1 might be influenced strongly by the combination of overtone from asymmetric C–O stretching and asymmetric N–O stretching. PLS regression models were built using these three wavelength ranges viz., 6430–6360 cm−1 , 5364–5290 cm−1 and 4849–4833 cm−1 . Two latent variables were used to make the PLS models. This number was used because of two reasons. One reason is that the TQ Analyst Software selected the two variables in its calculations, and the other reason is that it gave lower root mean squares error of cross validation (RMSECV). In order to find the closeness between reference value (value obtained through HPLC analysis) and the value found by the calibration model, RMSEP (root mean square error of prediction) was also calculated using the formula:
2.5. Sample preparation for nimesulide assay Plasma samples (275 l) were vigorously vortexed with the addition of acetonitrile (1000 l) in a microcentrifuge tube for the extraction of nimesulide; the tubes were then centrifuged at 1000 × g for 5 min and the solvent phases were separated in fresh microcentrifuge tubes. After the evaporation of the solvent phase, the samples were reconstituted in 100 l of acetonitrile which were then subjected to high-performance liquid chromatography (HPLC) analysis. A standard solution of nimesulide, in acetonitrile, was used for calibration of the HPLC peaks. 2.6. HPLC analysis For the calibration, as well as cross validation of PLS model developed using FT-NIR data, HPLC analysis were done as described by Toutain, et al. [15] and Rao, et al. [16], using an HPLC system with UV detector (LC 10A with SPD 10AT) from Shimadzu, Japan connected with a SymmetryShield (Waters) RP-18, column. The UV
RMSEP =
i
(Yipre − YiHPLC )
2
n
where n is the number of samples used for prediction. Similarly, root mean square error of calibration (RMSEC) and root mean square error of validation (RMSEV) were also calculated.
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159
Fig. 1. Raw FT-NIR spectra of plasma samples at different time interval, after the i.p. injection of nimesulide.
The PLS calibration model gave RMSEC of 2.11 ng, RMSEV of 2.89 ng and RMSEP of 2.54 ng. As the animal to animal variations is an influencing factor in the drug interaction, these values and variations are within the acceptable limits. Fig. 4 shows the calibration curve plotted using the concentration of nimesulide predicted using the FT-NIR method and the concentration of nimesulide measured using the HPLC method. The straight line curve showed a slope of 1.02 and an intercept of 0.79. The curve obtained from the PLS calibration model of the plasma samples fortified with nimesulide also gave comparable results with a straight line having regression equation 1.03x + 5.96 and r2 value of 0.99. The relative error was calculated using the equation: Error (%) =
YHPLC − Ypre × 100 YHPLC
where Y is the value of nimesulide content in the plasma. The plasma obtained after 5 min of the i.p. injection showed 100% error because the FT-NIR model developed failed to recognise the nimesulide related components in the spectra. There after the average relative error reduced to a range between −12.71% at 360 min and −1.83% at 60 min after the i.p. injection. The minus sign indicates
that the average predicted values of nimesulide (Ypre ) are numerically greater than that of the values obtained from HPLC analysis (YHPLC ). The percentage relative errors concerning the measurements are shown in Fig. 5. From this figure, it is possible to note that, in general, the relative errors decreased as the concentration of nimesulide increased in the rabbit plasma. The figure depicts a random distribution of the errors. The random distribution of relative errors is an indication of linear behaviour of the data [6]. Thus, it is clearly established that there is linearity in the measurements. If we consider all the 126 samples obtained in between 15 min and 480 min, then the average relative error is only −5.92%. Relative errors lower than ±6% are acceptable for quality control [20]. From the graph, it may be noted that most of the validation samples have errors of similar level. Paired t-test was also performed to check for any significant difference in the concentration of nimesulide in plasma samples predicted and determined using HPLC method. The t-value obtained was 0.47, and at the 95% confidence level, the critical value of t is 4.64 (p = 0.32). It can be concluded that the HPLC method and the PLS model developed using FT-NIR data do not significantly have different values for the determination of nimesulide in the plasma.
Fig. 2. FT-NIR secondary derivative spectra of plasma samples collected from nimesulide injected rabbits at different time after the i.p. injection. The wave number range 6430–6360 cm−1 .
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Table 1 PK Parameters obtained from the calculations using the raw data and PK87 software. Parameters
K(a) (min−1 ) Lambda (Z) (min−1 ) Absorbtion half time (min) Elimination half time (min) AUC (0 to tn) (ng min ml−1 ) AUC (0 to ∞) (ng min ml−1 ) AUMC (0 to tn) (ng min ml−1 ) AUMC (0 to ∞) (ng min ml−1 ) MRT (min) Clearance (ml min−1 ) VD (area) L C max (ng ml−1 ) C max calculated (ng ml−1 ) T max (min) T max calculated (min)
Using data from the HPLC analysis
Using data from the model (predicted)
Relative error percentage of means
Mean value (a)
STDEV
Mean value (b)
STDEV
[(a − b)/a] 100
0.0136 0.0089 50.77 77.98 18929.76 19518.65 3305768.82 3654867.30 187.27 10.25 69.24 75.29 70.01 60 83.56
0.0005 0.00032 1.29 2.76 560.53 551.55 108639.31 109909.11 3.18 0.29 3.63 6.65 3.04 0 2.28
−0.0133 −0.0089 52.03 78.20 19902.18 20547.26 3544892.25 3714492.75 191.17 9.74 65.88 76.62 71.24 60 84.11
0.00046 0.00076 1.36 7.33 655.66 694.52 138872.9 859841.6 5.69 0.33 5.83 7.22 2.65 0 2.81
The developed NIR model could not give correct measure of the nimesulide content in the plasma at 5 min readings. At 5 min, HPLC analysis indicated the presence of nimesulide in blood (0.64 ng ml−1 ). Thus, it can be assumed that the model is less sensitive than HPLC method, to the active component. This low sensitivity may be due to the preprocessing applied to the spectra. However, the model was clearly able to distinguish the samples with different concentrations of nimesulide in the blood samples collected after 5 min of i.p. injection. Hence, the further pharmacokinetics parameters of the nimesulide in rabbit were calculated, and the results are shown in Table 1. In both FT-NIR and HPLC analysis, it was clearly apparent that the plasma drug concentration continued to increase and reached a maximum at 60 min after the i.p. injection. There after, the drug concentration decreased gradually and reached a very low concentration at 480 min after the injection of the drug. Except at 5 min after the i.p. injection, the predicted values obtained from FT-NIR analysis were numerically closer to the values obtained from HPLC
2.21 0 −2.47 −0.28 −5.14 −5.27 −7.23 −1.63 −2.08 4.97 −4.86 −1.84 −1.76 0 0.01
serum analysis (Table 2). The nimesulide concentration in plasma vs. time curves showed quite similar trends in both FT-NIR based prediction and HPLC based analysis (Fig. 3) i.e., a rapid distribution phase and a slower elimination phase. Fig. 4 shows the calibration curve plotted using the concentration of nimesulide predicted using the NIR method and the concentration of nimesulide measured using the HPLC method. The drug concentration in plasma was highest (76.51 ± 7.38 ng ml−1 by FT-NIR method; 75.13 ± 6.81 ng ml−1 by HPLC method) at 60 min after i.p. injection and there after declined gradually and reached 5.62 ± 1.14 ng ml−1 by the FT-NIR based method (5.23 ± 0.29 ng ml−1 by HPLC measurement) at 480 min after i.p. (Table 2). The pharmacokinetic parameters like maximum concentration (C max), maximum concentration calculated (C max calc), maximum absorption time (T max), maximum absorption time calculated (T max calc), absorption half time, elimination half time, mean residence time, clearance rate, etc. obtained from both FT-NIR based PLS model analysis and HPLC
Table 2 Concentration of nimesulide in plasma at different times of observations in rabbits.
0.08 0.25 0.5 1 2 4 6 8
Predicted from our model
HPLC analysis
Mean value (ng ml−1 )
STDEV (ng ml−1 )
Mean value (ng ml−1 )
STDEV (ng ml−1 )
0 23.39 56.62 76.51 65.13 47.16 24.80 5.62
0 1.70 2.29 7.38 1.96 1.71 2.49 1.14
0.64 21.82 55.04 75.13 63.56 44.21 22.01 5.22
0.05 1.71 2.42 6.81 2.03 1.85 1.62 0.29
Amount of nimesulide (ng)
Time (h)
90 80 70 60 50
Predicted Value
40
HPLC value
30 20 10 0 0
50
100
150
200
250
300
350
400
450
Minutes Fig. 3. Time–concentration of nimesulide curve obtained for treated rabbits.
500
Values obtained by FT-NIR method (ng/ml)
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161
90 80
2
R = 0.9959
70 60 50 40 30 20 10 0 0
10
20
30
40
50
60
70
80
90
Values obtained by HPLC analysis (ng/ml) Fig. 4. Calibration curve for nimesulide content in the plasma samples obtained after i.p. injection.
120 100
% of error
80 60 40 20
Nimesulide concentration (ng/ml) in plasma
0 -20 0
10
20
30
40
50
60
70
80
90
-40 -60 Fig. 5. Relative error for nimesulide content obtained by the FT-NIR analysis.
based analysis were numerically close to each other (Table 1). There were no significant difference between the values obtained by the two methods (p > 0.05). Thus, it is clearly established that the PLS model developed using the FT-NIR data gave comparable results, in both the quantity of nimesulide present in blood plasma and the data obtained from pharmacokinetics study with those data, with HPLC method. The percentage of error difference between the two methods was less than 6%. Hence, the newly developed FT-NIR data based PLS model can be used in the pharmacokinetics and bio-availability study of nimesulide. Although methods like enzyme analysis, gas chromatography (GC) combined with or without mass spectrometry (MS), HPLC etc, are available to generate drug quantification data for the pharmacokinetics parameters and bio-availability studies, still many are depending on HPLC data. For all the routinely using methods, the extraction of the drug molecule from the blood plasma is required. In addition to the cost of solvents required for HPLC running, cost of solvents required for extraction and the process required are adding the total cost of analysis per samples as well as the time required for the analysis of one sample. In the method that we developed, there is no requirement of sample extraction, and thus no extra chemical is added to or subtracted from the plasma samples. Further, FT-NIR analysis does not require more than a minute to obtain the quantity of drug present in the sample. Thus, this method is much faster than the available HPLC methods and is environmental friendly. Most of the HPLC based protocols for nimesulide analysis available in the literature require 4.5–7.0 min to obtain the peak for nimesulide or its main metabolite 4-hydroxy nimesulide after injecting the sample into the instrument system. Our strategy is faster than the method reported by Ptácek et al. [21] in 2001. Their method can examine only 20 samples in an hour where as our method can give the result of more than 50 samples in an hour and is much more cost effective
than the other methods because no extraction, solvents and standards (after developing the model) are required. The latest in the queue, by Schebb et al., in 2011, reports [22] that an ultra fast online solid phase extraction liquid chromatography electrospray tandem mass spectrometry (SPE–LC–ESI–MS/MS) based method can determine diclofenac concentration in approximately 2 min rounding up to 30 samples in an hour. The FT-NIR based method is not only a high throughput analysis method but also provides further scope of automation and compliant with the requirements of green analytical chemistry. There are only two reports available in pharmacokinetics study of nimesulide in rabbits. In the first study, availability of nimesulide was examined, using HPLC, in aqueous humor after the administration of an ophthalmic preparation [23] and in the next one lithium content is noted after the co-administration of nimesulide and lithium [24]. There is no reports perce as far as pharmacokinetics study of nimesulide in the rabbit is concerned. To the best knowledge of the authors, there is no report of pharmacokinetics and bio-availability after i.p. administration of nimesulide to human or rabbits. The rabbit’s heart and cardiac circulation are very closely resemble those of human [17]. The T max calc obtained in the present study (84.11 ± 2.81 min) is in good agreement with T max of humans (73–165 min; 100 mg dosage), through the oral route [25], where as the elimination half life (78.2 ± 7.33 min) obtained for rabbit is less than that of human (108–284 min). 4. Conclusion This is the first report of demonstrating the capability of FT-NIR measurements in the pharmacokinetics and bio-availability studies. Here, we have performed in vivo measurements of nimesulide
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content in the plasma samples of rabbits after an i.p. injection in order to study kinetic behaviour of nimesulide. We have identified nimesulide-specific features in these in vivo spectra. The results suggest that FT-NIR transmission technique can be used for rapid and convenient analysis of drug content in plasma without any extraction procedure. The error of this method is within that expected range for pharmaceutical assays. The ease of use and rapid generation of results gives FT-NIR a distinct advantage over other analytical techniques. Since India is emerging as a centre of clinical research and pharmacovigilance, where a large number of drug exposed population is to be scanned, the FT-NIR method that have high speed and yet economical, may provide a viable alternate to the existing methods. Acknowledgement The authors are particularly thankful to Director, CSIR-CIMAP, Lucknow for his constant encouragement and giving facilities to achieve the findings. References [1] L. Stothers, B. Shadgan, A. Macnab, Urological application of near infrared spectroscopy, Can. J. Urol. 15 (2008) 4399–4409. [2] H. Azizian, J.K.G. Kramer, S.B. Heymsfield, Fourier transform near infrared spectroscopy: a newly developed: non-invasive method to measure body fat, Lipids 43 (2008) 97–103. [3] S. Takeno, T. Bamba, Y. Nakazawa, E.O.A. Fukusaki, A. Kobayashi, A highthroughput and solvent-free method for measurement of natural polyisoprene content in leaves by Fourier transform near infrared spectroscopy, J. Biosci. Bioeng. 106 (2008) 537–540. [4] M. Otsuka, I. Yamane, Prediction of tablet properties based on near infrared spectra of raw mixed powders by chemometrics: scale-up factor of blending and tableting processes, J. Pharm. Sci. 98 (2009) 4296–4305. [5] C. Abrahamsson, J. Johansson, S. Andersson-Engels, S.F.S. Svanberg, Time resolved NIR spectroscopy for quantitative analysis of intact pharmaceutical tablets, Anal. Chem. 77 (2005) 1055–1059. [6] W.F.C. Rocha, A.L. Rosa, J.A. Martins, R.J. Poppi, Determination and validation of nimesulide in pharmaceutical formulation by near infrared spectroscopy, J. Braz. Chem. Soc. 21 (2010) 1929–1936. [7] M.S. Kemper, E.J. Magnuson, S.R. Lowry, W.J. McCarthy, N. Aksornkoae, D.C. Watts, et al., Use of FT-NIR transmission spectroscopy for the quantitative analysis of an active ingredient in a translucent pharmaceutical topical gel formulation, AAPS PharmSci. 3 (2001), doi:10.1208/ps030323, article 23 (2001).
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
FT-NIR spectroscopy for rapid and simple determination of nimesulide in rabbit plasma for pharmacokinetic analysis P.V. Ajayakumar a,∗ , Debabrata Chanda b , Anirban Pal b , Mahendra P. Singh b , A. Samad c a
Analytical Chemistry Department, CSIR – Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India In-vivo Testing Facility, Molecular Bioprospection Department, CSIR – Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India c Microbiology and Plant pathology Department, CSIR – Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India b
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 19 September 2011 Accepted 19 September 2011 Available online 24 September 2011 Keywords: Pharmacokinetics Bio-availability FT-NIR Nimesulide Rabbit
a b s t r a c t High-throughput analysis of a large number of samples for pharmacokinetic study is necessary in drug development and pharmacovigilance. Usually, drug quantification for pharmacokinetics and bioavailability is achieved through matrix extraction and HPLC analysis, which is time, labour and cost intensive method. A prompt and solvent free method is the quest for such analysis in the present times. Pharmacokinetic analysis of nimesulide from plasma samples of rabbits through Fourier transform near infrared (FT-NIR) spectroscopy analysis combined with partial least squares (PLS) regression model was undertaken with validation through HPLC analysis. Pharmacokinetic parameters obtained through FTNIR and HPLC were found to be statistically similar with errors below the acceptable limits. The study demonstrates the use of FT-NIR for pharmacokinetics and bio-availability studies. This high throughput method analyses more than 50 samples in an hour without solvents usage and provide ample scope for automation and commercial utilization. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Near infrared (NIR) spectroscopy is a noninvasive, relatively low cost optical technique, portable, useful for real-time measurement of changes in tissues in relation to oxygenation and perfusion [1], body fat [2], qualitative and quantitative measurements of various chemicals in foods, pharmaceuticals, materials, medical, agricultural produce etc. [3]. In addition, Fourier transform near infrared (FT-NIR) is extensively used in different fields of the pharmaceutical industry like prediction of tablet properties of raw-mixed powders before compression [4]; quantitative measurement of components in intact tablets [5,6]; raw materials, intermediate and finished product forms including gels [7] and development of solid dosage forms [8] besides others. Recently, FT-NIR has extensively being explored for studies in urology [1,9]; sports medicine [10], realtime study of brain vascular and metabolic activities [11]. Much of the appeal of NIR technique is because of the fact that wealth of chemical and physical information can be obtained with in seconds often without the need for any sample preparation. Although Fourier transform infrared spectroscopy (FT-IR) has been used for determining molecular concentrations of different bio-active analytes in various biological matrices like blood, serum, plasma and urine [12], the use of FT-NIR in the bio-analysis of
pharmacophore in biological samples like blood, urine, plasma or serum samples has not yet been reported. There is a tremendous need and scope for novel and high throughput analytical method for bioanalysis in the course of drug discovery and development [13]. Hence in the present experiment, we have done pharmacokinetics study of nimesulide in rabbit through FT-NIR analysis of plasma samples and validated the same through HPLC analysis. Nimesulide (4-nitro-2-phenoxymethanesulfonanide) was chosen because of its continuing use in India and other developing countries as a prominent, selective cyclooxygenase (COX-2) inhibitor in inflammatory conditions as well as the easiest availability of high-performance liquid chromatography (HPLC) methods and IR data for the purpose of cross validation. The pharmacokinetics aspects of nimesulide have been documented in human [14] and some animal species using traditional methods [15,16], but none has explored the novel method of calculating kinetic parameters from FT-NIR generated data. Rabbits were preferred to other rodents because its heart and cardiac circulation are closely similar to those of human [17].
2. Materials and methods 2.1. Animals
∗ Corresponding author. Tel.: +91 522 2359632; fax: +91 522 2342666. E-mail address: [email protected] (P.V. Ajayakumar). 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.09.022
Adult male and female New Zealand white rabbits (2.5 kg body weight), six in numbers, being maintained at the in vivo testing
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facility, CIMAP, Lucknow, India were used for the study. The animals were acclimatized to the experimental environment for 7 days before the actual experimentation. The animals were re-used after a wash period of 21 days. The protocols used were duly approved by Institutional Animal Ethics Committee (IAEC) through CPCSEA, Government of India. 2.2. Chemicals Nimesulide and Cremophore EL were obtained from Sigma Chemicals, India. HPLC grade solvents were obtained from Merck India Ltd., India 2.3. Treatment of animals with nimesulide and collection of samples
detector was set at 210 nm and the analyses were carried out using the same series of the extracted plasma samples used in the FT-NIR analysis. 2.7. Kinetic analysis Pharmacokinetic parameters were calculated following the procedure as reported earlier [18] using the software PK87. The whole experiments were conducted three times in order to verify the reproducibility of the method. The spectra obtained from first and second rounds of experiments were used for calibration and validation of the PLS model. The spectra from the third round of the experiment were used only for prediction, and the predicted values were compared with the results from the HPLC analysis. 2.8. Calibration in raw plasma samples
A clear aqueous solution of nimesulide was prepared in 10% cremophore EL in 0.9% sodium chloride (NaCl) in water. Nimesulide was injected at 5 mg kg−1 body weight intraperitonially (i.p.) to the rabbits. Blood samples were collected in heparinised tubes, in duplicate, at 0, 5, 15, 30, 60, 120, 240, 360 and 480 min after the administration of the drug. Clear plasma samples were collected from the blood samples after centrifugation at 5000 × g for 5 min at 4 ◦ C.
A stock solution of nimesulide (5 mg ml−1 ) was prepared in double distilled water containing 0.9% NaCl and 12.5% cremophore EL. Two fold serial dilution of this nimesulide stock solution ranging from 2.5 mg ml−1 to 0.02 mg ml−1 was prepared in freshly collected rabbit plasma. Four replicates of the eight dilutions were subjected to FT-NIR analysis using plasma as blank.
2.4. FT-NIR analysis
3. Results and discussion
The plasma samples thus obtained were used directly for the Fourier transform near infrared (FT-NIR) analysis. The FT-NIR absorbance spectra from 10,000 cm−1 to 4000 cm−1 at a resolution of 4.0 cm−1 (using Antaris II Analyzer, Thermo Fisher Scientific, USA) in transmission mode, using indium gallium arsenide (InGaAs) detector were recorded for each sample. To reduce sampling error, the three vials (0.5 mm cell with 170 l rabbit plasma) of samples from each plasma sample prepared in the previous step were analysed individually. The plasma obtained from the blood collected at zero (0) minute (just before the administration of the drug) was considered as the blank and the background measurements were taken using the blank before every spectral measurement. Data acquisition, spectral mathematical treatments and partial least squares (PLS) regression were done using TQ Analyst Software (Thermo Fisher Scientific, USA). The second derivative of absorbance spectra smoothed with Norris derivative (segment length 19, gap between points 2) was used for all regression work. For path length corrections, standard normal variate (SNV) path length was chosen.
The raw absorbance spectra recorded for nimesulide in rabbit plasma, taken at different time intervals after the i.p. injection, are shown in Fig. 1. The FT-NIR data from first and second rounds of the experiment were randomly split into calibration set (64 samples, 4 rabbits) and validation set (32 samples, 2 rabbits). All the data from the third round of the experiment were used as prediction set (48 samples, 6 rabbits). The two validation animals were selected randomly in each time interval. The visual analysis of the second derivative spectra gave peaks and dips with an indication that at wave number ranges 6430–6360 cm−1 (Fig. 2), 5364–5290 cm−1 and 4849–4833 cm−1 the y-axis values change in the same way as the concentration of the drug, quantified through HPLC analysis, in the collected blood samples. At wave numbers ranging between 5364 and 5290 cm−1 , the spectra showed a dip. The FT-IR spectrum of the pure drug showed prominent peaks at wave numbers 3284, 1590, 1488, 1342, 1282 and 1247 cm−1 [19]. So the significant contribution of the FT-NIR band obtained at 6430–6360 cm−1 could be from the combination of overtones arised from N–H stretching and N–H deformation. Similarly, the major contribution for the band at 4849–4833 cm−1 might be influenced strongly by the combination of overtone from asymmetric C–O stretching and asymmetric N–O stretching. PLS regression models were built using these three wavelength ranges viz., 6430–6360 cm−1 , 5364–5290 cm−1 and 4849–4833 cm−1 . Two latent variables were used to make the PLS models. This number was used because of two reasons. One reason is that the TQ Analyst Software selected the two variables in its calculations, and the other reason is that it gave lower root mean squares error of cross validation (RMSECV). In order to find the closeness between reference value (value obtained through HPLC analysis) and the value found by the calibration model, RMSEP (root mean square error of prediction) was also calculated using the formula:
2.5. Sample preparation for nimesulide assay Plasma samples (275 l) were vigorously vortexed with the addition of acetonitrile (1000 l) in a microcentrifuge tube for the extraction of nimesulide; the tubes were then centrifuged at 1000 × g for 5 min and the solvent phases were separated in fresh microcentrifuge tubes. After the evaporation of the solvent phase, the samples were reconstituted in 100 l of acetonitrile which were then subjected to high-performance liquid chromatography (HPLC) analysis. A standard solution of nimesulide, in acetonitrile, was used for calibration of the HPLC peaks. 2.6. HPLC analysis For the calibration, as well as cross validation of PLS model developed using FT-NIR data, HPLC analysis were done as described by Toutain, et al. [15] and Rao, et al. [16], using an HPLC system with UV detector (LC 10A with SPD 10AT) from Shimadzu, Japan connected with a SymmetryShield (Waters) RP-18, column. The UV
RMSEP =
i
(Yipre − YiHPLC )
2
n
where n is the number of samples used for prediction. Similarly, root mean square error of calibration (RMSEC) and root mean square error of validation (RMSEV) were also calculated.
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Fig. 1. Raw FT-NIR spectra of plasma samples at different time interval, after the i.p. injection of nimesulide.
The PLS calibration model gave RMSEC of 2.11 ng, RMSEV of 2.89 ng and RMSEP of 2.54 ng. As the animal to animal variations is an influencing factor in the drug interaction, these values and variations are within the acceptable limits. Fig. 4 shows the calibration curve plotted using the concentration of nimesulide predicted using the FT-NIR method and the concentration of nimesulide measured using the HPLC method. The straight line curve showed a slope of 1.02 and an intercept of 0.79. The curve obtained from the PLS calibration model of the plasma samples fortified with nimesulide also gave comparable results with a straight line having regression equation 1.03x + 5.96 and r2 value of 0.99. The relative error was calculated using the equation: Error (%) =
YHPLC − Ypre × 100 YHPLC
where Y is the value of nimesulide content in the plasma. The plasma obtained after 5 min of the i.p. injection showed 100% error because the FT-NIR model developed failed to recognise the nimesulide related components in the spectra. There after the average relative error reduced to a range between −12.71% at 360 min and −1.83% at 60 min after the i.p. injection. The minus sign indicates
that the average predicted values of nimesulide (Ypre ) are numerically greater than that of the values obtained from HPLC analysis (YHPLC ). The percentage relative errors concerning the measurements are shown in Fig. 5. From this figure, it is possible to note that, in general, the relative errors decreased as the concentration of nimesulide increased in the rabbit plasma. The figure depicts a random distribution of the errors. The random distribution of relative errors is an indication of linear behaviour of the data [6]. Thus, it is clearly established that there is linearity in the measurements. If we consider all the 126 samples obtained in between 15 min and 480 min, then the average relative error is only −5.92%. Relative errors lower than ±6% are acceptable for quality control [20]. From the graph, it may be noted that most of the validation samples have errors of similar level. Paired t-test was also performed to check for any significant difference in the concentration of nimesulide in plasma samples predicted and determined using HPLC method. The t-value obtained was 0.47, and at the 95% confidence level, the critical value of t is 4.64 (p = 0.32). It can be concluded that the HPLC method and the PLS model developed using FT-NIR data do not significantly have different values for the determination of nimesulide in the plasma.
Fig. 2. FT-NIR secondary derivative spectra of plasma samples collected from nimesulide injected rabbits at different time after the i.p. injection. The wave number range 6430–6360 cm−1 .
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Table 1 PK Parameters obtained from the calculations using the raw data and PK87 software. Parameters
K(a) (min−1 ) Lambda (Z) (min−1 ) Absorbtion half time (min) Elimination half time (min) AUC (0 to tn) (ng min ml−1 ) AUC (0 to ∞) (ng min ml−1 ) AUMC (0 to tn) (ng min ml−1 ) AUMC (0 to ∞) (ng min ml−1 ) MRT (min) Clearance (ml min−1 ) VD (area) L C max (ng ml−1 ) C max calculated (ng ml−1 ) T max (min) T max calculated (min)
Using data from the HPLC analysis
Using data from the model (predicted)
Relative error percentage of means
Mean value (a)
STDEV
Mean value (b)
STDEV
[(a − b)/a] 100
0.0136 0.0089 50.77 77.98 18929.76 19518.65 3305768.82 3654867.30 187.27 10.25 69.24 75.29 70.01 60 83.56
0.0005 0.00032 1.29 2.76 560.53 551.55 108639.31 109909.11 3.18 0.29 3.63 6.65 3.04 0 2.28
−0.0133 −0.0089 52.03 78.20 19902.18 20547.26 3544892.25 3714492.75 191.17 9.74 65.88 76.62 71.24 60 84.11
0.00046 0.00076 1.36 7.33 655.66 694.52 138872.9 859841.6 5.69 0.33 5.83 7.22 2.65 0 2.81
The developed NIR model could not give correct measure of the nimesulide content in the plasma at 5 min readings. At 5 min, HPLC analysis indicated the presence of nimesulide in blood (0.64 ng ml−1 ). Thus, it can be assumed that the model is less sensitive than HPLC method, to the active component. This low sensitivity may be due to the preprocessing applied to the spectra. However, the model was clearly able to distinguish the samples with different concentrations of nimesulide in the blood samples collected after 5 min of i.p. injection. Hence, the further pharmacokinetics parameters of the nimesulide in rabbit were calculated, and the results are shown in Table 1. In both FT-NIR and HPLC analysis, it was clearly apparent that the plasma drug concentration continued to increase and reached a maximum at 60 min after the i.p. injection. There after, the drug concentration decreased gradually and reached a very low concentration at 480 min after the injection of the drug. Except at 5 min after the i.p. injection, the predicted values obtained from FT-NIR analysis were numerically closer to the values obtained from HPLC
2.21 0 −2.47 −0.28 −5.14 −5.27 −7.23 −1.63 −2.08 4.97 −4.86 −1.84 −1.76 0 0.01
serum analysis (Table 2). The nimesulide concentration in plasma vs. time curves showed quite similar trends in both FT-NIR based prediction and HPLC based analysis (Fig. 3) i.e., a rapid distribution phase and a slower elimination phase. Fig. 4 shows the calibration curve plotted using the concentration of nimesulide predicted using the NIR method and the concentration of nimesulide measured using the HPLC method. The drug concentration in plasma was highest (76.51 ± 7.38 ng ml−1 by FT-NIR method; 75.13 ± 6.81 ng ml−1 by HPLC method) at 60 min after i.p. injection and there after declined gradually and reached 5.62 ± 1.14 ng ml−1 by the FT-NIR based method (5.23 ± 0.29 ng ml−1 by HPLC measurement) at 480 min after i.p. (Table 2). The pharmacokinetic parameters like maximum concentration (C max), maximum concentration calculated (C max calc), maximum absorption time (T max), maximum absorption time calculated (T max calc), absorption half time, elimination half time, mean residence time, clearance rate, etc. obtained from both FT-NIR based PLS model analysis and HPLC
Table 2 Concentration of nimesulide in plasma at different times of observations in rabbits.
0.08 0.25 0.5 1 2 4 6 8
Predicted from our model
HPLC analysis
Mean value (ng ml−1 )
STDEV (ng ml−1 )
Mean value (ng ml−1 )
STDEV (ng ml−1 )
0 23.39 56.62 76.51 65.13 47.16 24.80 5.62
0 1.70 2.29 7.38 1.96 1.71 2.49 1.14
0.64 21.82 55.04 75.13 63.56 44.21 22.01 5.22
0.05 1.71 2.42 6.81 2.03 1.85 1.62 0.29
Amount of nimesulide (ng)
Time (h)
90 80 70 60 50
Predicted Value
40
HPLC value
30 20 10 0 0
50
100
150
200
250
300
350
400
450
Minutes Fig. 3. Time–concentration of nimesulide curve obtained for treated rabbits.
500
Values obtained by FT-NIR method (ng/ml)
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161
90 80
2
R = 0.9959
70 60 50 40 30 20 10 0 0
10
20
30
40
50
60
70
80
90
Values obtained by HPLC analysis (ng/ml) Fig. 4. Calibration curve for nimesulide content in the plasma samples obtained after i.p. injection.
120 100
% of error
80 60 40 20
Nimesulide concentration (ng/ml) in plasma
0 -20 0
10
20
30
40
50
60
70
80
90
-40 -60 Fig. 5. Relative error for nimesulide content obtained by the FT-NIR analysis.
based analysis were numerically close to each other (Table 1). There were no significant difference between the values obtained by the two methods (p > 0.05). Thus, it is clearly established that the PLS model developed using the FT-NIR data gave comparable results, in both the quantity of nimesulide present in blood plasma and the data obtained from pharmacokinetics study with those data, with HPLC method. The percentage of error difference between the two methods was less than 6%. Hence, the newly developed FT-NIR data based PLS model can be used in the pharmacokinetics and bio-availability study of nimesulide. Although methods like enzyme analysis, gas chromatography (GC) combined with or without mass spectrometry (MS), HPLC etc, are available to generate drug quantification data for the pharmacokinetics parameters and bio-availability studies, still many are depending on HPLC data. For all the routinely using methods, the extraction of the drug molecule from the blood plasma is required. In addition to the cost of solvents required for HPLC running, cost of solvents required for extraction and the process required are adding the total cost of analysis per samples as well as the time required for the analysis of one sample. In the method that we developed, there is no requirement of sample extraction, and thus no extra chemical is added to or subtracted from the plasma samples. Further, FT-NIR analysis does not require more than a minute to obtain the quantity of drug present in the sample. Thus, this method is much faster than the available HPLC methods and is environmental friendly. Most of the HPLC based protocols for nimesulide analysis available in the literature require 4.5–7.0 min to obtain the peak for nimesulide or its main metabolite 4-hydroxy nimesulide after injecting the sample into the instrument system. Our strategy is faster than the method reported by Ptácek et al. [21] in 2001. Their method can examine only 20 samples in an hour where as our method can give the result of more than 50 samples in an hour and is much more cost effective
than the other methods because no extraction, solvents and standards (after developing the model) are required. The latest in the queue, by Schebb et al., in 2011, reports [22] that an ultra fast online solid phase extraction liquid chromatography electrospray tandem mass spectrometry (SPE–LC–ESI–MS/MS) based method can determine diclofenac concentration in approximately 2 min rounding up to 30 samples in an hour. The FT-NIR based method is not only a high throughput analysis method but also provides further scope of automation and compliant with the requirements of green analytical chemistry. There are only two reports available in pharmacokinetics study of nimesulide in rabbits. In the first study, availability of nimesulide was examined, using HPLC, in aqueous humor after the administration of an ophthalmic preparation [23] and in the next one lithium content is noted after the co-administration of nimesulide and lithium [24]. There is no reports perce as far as pharmacokinetics study of nimesulide in the rabbit is concerned. To the best knowledge of the authors, there is no report of pharmacokinetics and bio-availability after i.p. administration of nimesulide to human or rabbits. The rabbit’s heart and cardiac circulation are very closely resemble those of human [17]. The T max calc obtained in the present study (84.11 ± 2.81 min) is in good agreement with T max of humans (73–165 min; 100 mg dosage), through the oral route [25], where as the elimination half life (78.2 ± 7.33 min) obtained for rabbit is less than that of human (108–284 min). 4. Conclusion This is the first report of demonstrating the capability of FT-NIR measurements in the pharmacokinetics and bio-availability studies. Here, we have performed in vivo measurements of nimesulide
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content in the plasma samples of rabbits after an i.p. injection in order to study kinetic behaviour of nimesulide. We have identified nimesulide-specific features in these in vivo spectra. The results suggest that FT-NIR transmission technique can be used for rapid and convenient analysis of drug content in plasma without any extraction procedure. The error of this method is within that expected range for pharmaceutical assays. The ease of use and rapid generation of results gives FT-NIR a distinct advantage over other analytical techniques. Since India is emerging as a centre of clinical research and pharmacovigilance, where a large number of drug exposed population is to be scanned, the FT-NIR method that have high speed and yet economical, may provide a viable alternate to the existing methods. Acknowledgement The authors are particularly thankful to Director, CSIR-CIMAP, Lucknow for his constant encouragement and giving facilities to achieve the findings. References [1] L. Stothers, B. Shadgan, A. Macnab, Urological application of near infrared spectroscopy, Can. J. Urol. 15 (2008) 4399–4409. [2] H. Azizian, J.K.G. Kramer, S.B. Heymsfield, Fourier transform near infrared spectroscopy: a newly developed: non-invasive method to measure body fat, Lipids 43 (2008) 97–103. [3] S. Takeno, T. Bamba, Y. Nakazawa, E.O.A. Fukusaki, A. Kobayashi, A highthroughput and solvent-free method for measurement of natural polyisoprene content in leaves by Fourier transform near infrared spectroscopy, J. Biosci. Bioeng. 106 (2008) 537–540. [4] M. Otsuka, I. Yamane, Prediction of tablet properties based on near infrared spectra of raw mixed powders by chemometrics: scale-up factor of blending and tableting processes, J. Pharm. Sci. 98 (2009) 4296–4305. [5] C. Abrahamsson, J. Johansson, S. Andersson-Engels, S.F.S. Svanberg, Time resolved NIR spectroscopy for quantitative analysis of intact pharmaceutical tablets, Anal. Chem. 77 (2005) 1055–1059. [6] W.F.C. Rocha, A.L. Rosa, J.A. Martins, R.J. Poppi, Determination and validation of nimesulide in pharmaceutical formulation by near infrared spectroscopy, J. Braz. Chem. Soc. 21 (2010) 1929–1936. [7] M.S. Kemper, E.J. Magnuson, S.R. Lowry, W.J. McCarthy, N. Aksornkoae, D.C. Watts, et al., Use of FT-NIR transmission spectroscopy for the quantitative analysis of an active ingredient in a translucent pharmaceutical topical gel formulation, AAPS PharmSci. 3 (2001), doi:10.1208/ps030323, article 23 (2001).
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