A rapid quantitative assay of intact paracetamol tablets by reflectance near-infrared spectroscopy Andrew D. Trafford,*a Roger D. Jee,a Anthony C. Moffata and Paul Grahamb a Centre for Pharmaceutical Analysis, School of Pharmacy, University of London,29/39 Brunswick Square, London, UK WC1N 1AXb Sanofi Research Division, Alnwick Research Centre, Willowburn Avenue, Alnwick,Northumberland, UK NE66 2JHReceived 24th August 1998, Accepted 3rd December 1998
Near-infrared (NIR) reflectance spectroscopy was used to determine rapidly and non-destructively the content ofparacetamol in bulk batches of intact Sterwin 500 mg tablets by collecting NIR spectra in the range1100–2500 nm and using a multiple linear regression calibration method. The developed NIR method gave resultscomparable to the British Pharmacopoeia 1993 UV assay procedure, the standard errors of calibration andprediction being 0.48% and 0.71% m/m, respectively. The method showed good repeatability, the standarddeviation and coefficient of variation for six NIR assays on the same batch on the same day being 0.14 and0.16% m/m, respectively, while measurements over six consecutive days gave 0.31 and 0.36% m/m, respectively. Applying the calibration to a parallel test set gave a mean bias of 20.22% and a mean accuracy of 0.45%. Thedeveloped method illustrates how the full potential of NIR can be utilised and how the ICH guidelines whichrecommend the validation of linearity, range, accuracy and precision for pharmaceutical registration purposes canbe applied. Duplicate determinations on bulk batches could be performed in under 2 min, allowing the potentialuse of the method on-line for real time monitoring of a running production process.
Near-infrared (NIR) absorption is mainly due to overtone and
sation (ICH) guidelines12 to the assay. The ICH guidelines
combination vibrations arising from fundamental vibrations in
recommend the validation of linearity, range, accuracy and
the mid-infrared region. As these absorptions are weak, NIR
precision, both short and long term, for analytical procedures
spectroscopy should be ideally suited for the analysis of intact
which are to be used for pharmaceutical registration purposes.
tablets. No sample preparation is required and the method isnon-destructive. Surprisingly, the majority of published appli-cations of NIR spectroscopy to tablets have not exploited this
Experimental
advantage, but used powdered tablets or solutions. Zappala andPost1 measured meprobamate in tablets after extraction into
Materials
chloroform and ranitidine chlorohydrate,2 ranitidine hydro-chloride3 and cefuroxime acetil4 have all been measured as
Forty-five batches of paracetamol tablets were used for the
powdered tablet samples. An early example of an intact tablet
study; 35 were normal production batches of Sterwin 500 mg
analysis was a qualitative study5 to identify adulterated aspirin
paracetamol tablets (nominal content 84.175% m/m para-
tablets. Quantitative assays based on intact tablets have
cetamol) with a nominal diameter of 12.7 mm and thickness of
included the assay of amiodarone chlorohydrate,6 metoprolol,7
3.80–4.15 mm and 10 were development batches (76–92% m/m
SB 216469-S8 and aspirin.9 Recently, we described the
paracetamol, i.e., 90–110% of the nominal label content). The
application of transmittance NIR spectroscopy to the analysis of
development batches were of the same dimensions as the
individual intact tablets10,11 and compared the effect of various
normal production batches but were specially manufactured for
pre-treatments on partial least-squares regression models;
this study at a pilot scale on a small scale press, by altering the
experiments involved the measurement of 20 intact tablets with
content of paracetamol and the major excipient (starch). The
an analysis time of 10 min per batch.
raw materials used were Paracetamol Fine PhEur, Potassium
A problem with intact tablet assays is that normal production
Sorbate PhEur, Povidone K-25 PhEur, Pregelatinised Starch
batches do not encompass a sufficiently wide range for setting
USNF, Starch Maize PhEur, Stearic Acid BP and Sterilised Talc
up a reliable calibration equation. Tablets which are both under-
PhEur, which were of the same specification as those used in the
and overdosed with respect to the analyte need to be produced
tablets and were obtained from Sanofi Research Division,
without altering other factors that may affect the assay such as
Alnwick Research Centre, Alnwick, Northumberland, UK.
particle size, moisture content and compaction characteristics.
All reagents were of analytical-reagent grade and were
Although this complicates the calibration, as compared with
obtained from BDH (Poole, Dorset, UK).
powdered tablet assays for which it is easy to prepare standards,the long-term gains of no sample preparation and the potentialfor the use on-line, allowing for real time monitoring of a
UV analysis
running production process, make the effort worthwhile.
This paper describes a rapid quantitative assay for the
UV measurements were made using a Perkin-Elmer (Beacons-
determination of paracetamol in bulk batches of intact tablets
field, Buckinghamshire, UK) Lambda 15 UV/VIS spec-
using diffuse reflectance NIR spectroscopy and multiple
trophotometer and 1 cm pathlength matched silica cells. Tablet
wavelength linear regression. Where possible we applied the
batches were assayed in duplicate using the following procedure
recommendations of the International Conference on Harmoni-
based on the British Pharmacopoeia 199313 assay. The
Analyst, 1999, 124, 163–167
Pharmacopoeia method was modified because out of specifica-
Reference analysis
tion tablets were being analysed. For the assay, 20 tablets wereground to a fine powder and accurately 0.15 g were measured
Reference values (‘true’ values) for the paracetamol content of
and transferred into a 200.0 ml calibrated flask. A 50 ml volume
all batches of tablets were determined in duplicate using the UV
of 0.1 m sodium hydroxide solution was added and the sample
assay procedure. The precision of the UV assay procedure was
was shaken for 10 min, then 100 ml of distilled water were
estimated by pooling the results from the duplicate measure-
added and the sample shaken for a further 10 min. The solution
ments made on the 35 calibration and validation batches and
was diluted to volume with distilled water, mixed and filtered
calculating the standard deviation (s) using the equation
and 5.0 ml of the solution were pipetted into a 500.0 ml
calibrated flask. 50 ml of 0.1 m sodium hydroxide solution were
added and the solution made to volume with distilled water. The
absorbance was measured at 257 nm using 0.01 m sodium
hydroxide solution as a blank. The content of paracetamol was
calculated taking 715 as the value of A1% at the maximum at
Where n is the number of duplicates, k1 and k2 are the individualduplicate results and ¯xk is the mean of the duplicates. Thestandard deviation was 0.31% m/m, giving a standard error (SE)
NIR analysis
for a UV determined reference value of 0.22% m/m using theequation
NIR reflectance spectra were measured using a NIRSystems
6500 near-infrared spectrophotometer (Foss NIRSystems,
Maidenhead, Berkshire, UK) fitted with a reflectance detector(NR-6503), sample transport module (NR-6511) and coarsesample cell (NR-7080). The instrument was governed by NSAS(Near-Infrared Spectral Analyses Software) version 3.52 (Foss
Method development and calibration
NIRSystems). Each measured spectrum was the average of 32scans and measured over the wavelength range 1100–2500 nm.
The NIR spectra were transferred to the Vision software along
Batches were measured by pouring approximately 100 tablets
with the mean UV assay values for the paracetamol content of
into the coarse sample cell and scanning on the quarter full
each batch. Absorbance spectra were treated mathematically by
setting. Measurements were made in duplicate, the coarse
performing a standard normal variate (SNV) transformation14 to
sample cell being refilled with the same tablets between scans.
remove mutiplicative interferences of scatter and particle size
Spectra were processed using Vision beta software (Foss
effectively, followed by calculating the second derivative
(segment size of 20 and a gap size of zero data points) tomaintain the peak locations but enhance the resolution. Fifteennormal production batches were assigned to a calibration set
Results and discussion
and 10 to a validation set and forward search multiple linearregression (MLR), using the Vision program, was applied to the
Feasibility study
data. A poor multiple correlation coefficient (R2) between theUV and NIR values of 0.694 was obtained at 1926 nm, which
NIR reflectance spectra of all the tablet ingredients were
only improved to R2 = 0.762 when a second wavelength was
recorded as log (1/R), where R is the reflectance, and their
added. This suggested that it was not possible to generate
second derivative absorbance spectra were examined to identify
acceptable calibration equations using only normal production
any unique spectral features of the active constituent (para-
batches owing to the limited range of paracetamol concentra-
cetamol). The second derivative spectrum of paracetamol
tions in normal production batches. To improve the calibration
showed characteristic spectral features at about 1525 and
equation, five development batches, which covered the ex-
1625–1675 nm; the minimum at about 1525 nm appeared to be
tremes of concentration, were added to the calibration set and a
the least affected by peaks from the excipients. When the second
further five development batches added to the validation set.
derivative spectra of three development batches (76, 84 and
Forward search MLR gave R2 = 0.971 at 1426 nm, which
93% m/m paracetamol, equivalent to 90, 100 and 110% of the
improved to 0.974 when a second wavelength, 1528 nm, was
nominal tablet content, respectively; Fig. 1) were compared,
added. This second wavelength corresponded to the character-
they had a minimum in this wavelength region which correlated
istic spectral feature of paracetamol identified in the feasibility
well with the paracetamol content in the tablets.
When the first wavelength was selected manually from the
characteristic spectral region, 1530 nm gave the best correlationwith R2 = 0.860. When a second wavelength of 1426 nm,selected by the Vision software, was added, R2 improved to0.974, the same as the initial two wavelength MLR selected bythe Vision software. On addition of a third wavelength, R2 onlyimproved to 0.976, a minor improvement considering thesubsequent risk of overfitting the data.
The two wavelength calibration was therefore chosen and the
Y = 87.22 2 35.11A1530 + 169.65A1426
where Y is the predicted paracetamol content (% m/m) and A1530and A1426 are the ordinate values of the transformed spectra(SNV plus the second derivative) at 1530 and 1426 nm,respectively. The fit had a residual sum of squares (RSS) of 3.89
Second derivative absorbance NIR spectra of intact paracetamol
[eqn. (4)], giving a standard error of calibration (SEC) of 0.48%
tablets: (a) 76, (b) 84 and (c) 93% m/m paracetamol content. Analyst, 1999, 124, 163–167 versus UV assay values is shown in Fig. 3. Again, the 95%
confidence intervals for the intercept (211.52 to 11.92) and
slope (0.93–1.07) suggest that there is no evidence for relative
Parallel test set
where y is the ‘true’ paracetamol content (i.e., mean UV assayvalue), n the number of batches and p the number of coefficients
Further validation was performed on batches which were
in eqn. (3). Fig. 2 shows a plot of predicted values versus
independent of both the calibration and validation sets.15 The
reference values for the calibration set. Ideally, the intercept (a)
validation set used above was not totally independent of the
and slope (b) should be 0 and 1, respectively, if there is no fixed
calibration set as they were scanned at the same time. Validation
systematic error or relative systematic error in the calibration
batches are often used to tune a calibration procedure by, for
equation. Linear regression was applied and the 95% con-
example, choosing optimum wavelengths for a multiple regres-
fidence intervals for the intercept [eqn. (6)] and slope [eqn. (7)]
sion equation so the use of a parallel test set avoids overfitting.
Also the performance of the calibration on the parallel test set isa more reliable indicator of its future performance on normal
production batches. A month after the calibration and validation
sets had been measured, 10 totally independent production
batches were used to evaluate the performance of the calibra-
tion. The UV and NIR results (Table 1) were then compared
using the paired sampled Student’s t-test [eqn. (10)] to show ifthe results were statistically equivalent.
where t is Student’s t at the 95% probability level and n 2 2degrees of freedom, RSD is the residual standard deviation
xx is the sum of squares [eqn. (9)].
where d is the mean residual (NIR 2 UV) and sd is the standard
deviation of the residuals. A value of 1.069 for tcalc wasobtained. The two-sided critical value of Students’ t at the 5%
significance level for n 2 2 degrees of freedom was 2.262,
suggesting that there was no evidence for a difference between
The confidence interval for the intercept (23.56 to 8.08)
included 0, and there was therefore no evidence to suggest anon-zero intercept. Similarly, the confidence interval for theslope (0.90–1.04) included 1, suggesting that there was no
Accuracy
evidence for a relative systematic error in the calibrationequation.
For the purposes of this study, the accuracy was taken as howclose the NIR values were to the UV assay values. The standarderrors of calibration (0.48% m/m) and prediction (0.71% m/m)
Validation of calibration equation
gave an indication of the accuracy of the NIR determination anda further estimate of accuracy was given by the standard
The calibration equation was validated by using it to calculate
deviation of the residuals obtained in the parallel test set (0.53%
the paracetamol content of each of the batches in the validation
m/m). As expected, these values were greater than the standard
sample set. The standard error for prediction (SEP) was 0.71%
error (0.22% m/m) for the UV measurement itself, although
m/m (equation as for SEC with p = 0). A plot of predicted
acceptable and therefore suitable for batch release purposes.
Plot of NIR predicted versus UV determined paracetamol content
Plot of NIR predicted versus UV determined paracetamol content
(% m/m) for the calibration set (r = 0.989).
(% m/m) for the validation set (r = 0.962). Analyst, 1999, 124, 163–167
The mean bias [eqn. (11)] and the mean accuracy [eqn. (12)] for
the parallel test set (n = 10) were determined to be 20.22 and0.45% with standard deviations of 0.63 and 0.48%, re-
NIR determinations of paracetamol content (% m/m)
there was no evidence that the analysts were not equallyprecise. Repeatability
The short term precision (within-day) was determined bymeasuring the paracetamol content of a single batch six times
Conclusions
within one day by both UV and NIR methods. The standarddeviation and coefficient of variation (CV) for the NIR assay
The developed one step NIR method for the quantitative assay
procedure were 0.14% m/m and 0.16%, respectively, and for the
of paracetamol in intact Sterwin 500 mg tablets was rapid and
UV assay procedure they were 0.41% m/m and 0.48%,
statistical analysis of the data indicated that the NIR method was
respectively. The mean (±95% confidence limit) UV and NIR
comparable to the British Pharmacopoeia 1993 reference
assay values were 84.53 ± 0.43% and 84.38 ± 0.14% m/m,
respectively. The confidence intervals overlap, further suggest-
The NIR procedure had the advantages over the reference
ing that there was no evidence for a difference in values
technique of requiring no sample preparation or the use of
obtained by the two procedures (cf., parallel test set).
potential environmentally harmful reagents. Although the ICHguidelines were developed mainly for the validation of
Intermediate precision
analytical procedures primarily based on the analyte being insolution, it was found possible to apply them successfully to the
The between-day precision was measured by assaying a single
validation of a reflectance NIR intact bulk tablet assay. This
batch on six consecutive days by both UV and NIR methods.
paper illustrates how the full potential of NIR can be utilised and
The standard deviation and CV for the NIR assay procedure
the ICH guidelines applied to the quantification of active
were 0.31% m/m and 0.36%, respectively, and for the UV assay
ingredients in bulk samples of intact tablets and it is hoped that
procedure they were 0.52% m/m and 0.61%, respectively. As in
this will lead to others developing rapid tablet assays. Although
the repeatability study, the mean (±95% confidence limit)
the calibration process requires the preparation of intact tablets
UV and NIR results, 84.52 ± 0.54% m/m and 84.15 ± 0.32%
covering the calibration range and analyses by a reference
m/m, respectively, gave no evidence for a difference in the
method, once the calibration has been established duplicate
quantitative determinations on bulk batches can be performed in
The between analyst precision with the NIR procedure was
determined by six different analysts testing the same batch six
The mean bias (20.22%) and mean accuracy (0.45%) were
times on the same day (Table 2). Reference scans were taken
comparable to those for the assay on individual intact tablets by
between the different analysts’ scans and the coarse sample cell
transmission NIR spectroscopy,10 which had values of 20.08%
was refilled between each scan. The hypothesis that all the
and 0.59%, respectively, but was five times quicker and
analysts’ results were equally precise was tested using Co-
therefore ideally suited to the analysis of bulk tablets on-line,
chran’s test.16 This test compares the largest variance (s2) with
allowing real time monitoring of a running production proc-
the other variances by dividing the largest variance by the sum
of all the variances. This was determined to be 0.413, which was
A method similar to the above using a sample transport
less than the critical value of 0.445 (5% significance level), so
module, coarse sample cell and a reflectance detector has been
Analyst, 1999, 124, 163–167
accepted by the Medicines Control Agency for the assay of
S. M. Han and P. G. Faulkner, J. Pharm. Biomed. Anal., 1996, 14,
paracetamol in intact Sterwin tablets.
P. Merkle and K. A. Kovar, J. Pharm. Biomed. Anal., 1997, 17,
The authors thank Foss NIRSystems for the loan of the
NIRSystems 6500 spectrophotometer and Sanofi, Eli Lilly and
A. Eustaquio, P. Graham, R. D. Jee, A. C. Moffat and A. D. Trafford,
Bristol-Myers Squibb for funding a research studentship. Analyst, 1998, 123, 2303.
M. Blanco, A. Eustaquio, R. D. Jee and A. C. Moffat, Anal. Chim. Acta, in the press.
International Conference on Harmonisation (ICH) of Technical
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