PLEASE SCROLL DOWN FOR ARTICLE

This article was downloaded by: [Bunaciu, Andrei]On: 18 May 2010Access details: Access Details: [subscription number 922356051]Publisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Analytical LettersPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713597227

Estimation of Uncertainty for Measuring Codeine Phosphate TabletsFormulation Using UV-Vis SpectrophotometryIoana Diaconu a; Hassan Y. Aboul-Enein bc; Andrei A. Bunaciu d;Ion G. Tănase e

a Faculty of Applied Chemistry and Materials Science, Department of Analytical Chemistry andInstrumental Analysis, University Politehnica Bucharest, Bucharest, Romania b Pharmaceutical andMedicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, NationalResearch Centre, Cairo, Egypt c Faculty of Science, Department of Chemistry, Universsiti TeknologiMalaysia, UTM Skudai, Johor, Malaysia d CROMATEC_PLUS SRL, Analytical Research Department,Bucharest, Romania e Faculty of Chemistry, Department of Analytical Chemistry, University ofBucharest, Bucharest, Romania

Online publication date: 18 May 2010

To cite this Article Diaconu, Ioana , Aboul-Enein, Hassan Y. , Bunaciu, Andrei A. andTănase, Ion G.(2010) 'Estimation ofUncertainty for Measuring Codeine Phosphate Tablets Formulation Using UV-Vis Spectrophotometry', AnalyticalLetters, 43: 7, 1207 — 1216To link to this Article: DOI: 10.1080/00032710903518633URL: http://dx.doi.org/10.1080/00032710903518633

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.

Spectrophotometry

ESTIMATION OF UNCERTAINTY FOR MEASURINGCODEINE PHOSPHATE TABLETS FORMULATIONUSING UV-VIS SPECTROPHOTOMETRY

Ioana Diaconu,1 Hassan Y. Aboul-Enein,2,3

Andrei A. Bunaciu,4 and Ion G. Tanase51Faculty of Applied Chemistry and Materials Science, Department ofAnalytical Chemistry and Instrumental Analysis, University PolitehnicaBucharest, Bucharest, Romania2Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical andDrug Industries Research Division, National Research Centre, Cairo, Egypt3Faculty of Science, Department of Chemistry, Universsiti TeknologiMalaysia, UTM Skudai, Johor, Malaysia4CROMATEC_PLUS SRL, Analytical Research Department,Bucharest, Romania5Faculty of Chemistry, Department of Analytical Chemistry, University ofBucharest, Bucharest, Romania

Analytical results represent a very important part in a quality control program. Uncertainty

estimation is an important step in method validation. The objective of this paper is to study the

uncertainty of measurement estimation in the quantitative determination of codeine phosphate

from pharmaceutical formulations using UV-VIS spectrophotometry. The uncertainty esti-

mation was performed using the Ishikawa diagram. The estimation of uncertainty components

proved to be a good way for the experimental model to obtain low contribution of uncertainty

to the analytical result.

Keywords: Codeine analysis; Uncertainty estimation; UV-Vis spectrophotometry; Validation

INTRODUCTION

Codeine (Codeine Phosphate Hemihydrate, (5R,6S)-7,8-didehydro-4,5-epoxy-3-methoxy-N-methylmorphinan-6-ol phosphate hemihydrate; Fig. 1), is an opiatethat is used for its analgesic and antitussive properties (European Pharmacopoeia,

Received 4 October 2009; accepted 30 November 2009.

This paper was submitted as part of a Special Memorial Issue honoring George-Emil Baiulescu.

Address correspondence to Professor Hassan Y. Aboul-Enein, Pharmaceutical and Medicinal

Chemistry Department, Pharmaceutical and Drug Industries Research Division, Dokki, Cairo 12311,

Egypt. E-mail: enein@gawab.com

Analytical Letters, 43: 1207–1216, 2010

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032710903518633

1207

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

2008). It is one of the most used pharmaceutical drugs according to numerousreports of World Health Organization (WHO).

Literature data has presented several methods for the determination of codeineutilizing several methods, such as: gas chromatography (GC) (Papoutsis et al. 2008),high performance liquid chromatography (HPLC) (Pascual and Sanagustın 1999),gas chromatography-mass spectrometry (GC-MS) (Lachenmeier, Musshoff, andMadea 2006), thin layer chromatography (TLC) (Dimitrovskat et al. 1995), andcapillary electrophoresis (CE) (Zhang et al. 2007; Hindson et al. 2007).

Reliable analytical methods are needed in all the fields of chemical analysis inorder to achieve precise results. A measurement or a test is complete only if itincludes an uncertainty estimation test.

There are some papers that propose the estimation of analytical measurementsuncertainty (CITAC=EURACHEM 2000; ISO Guide 1995; Tanase et al. 2006;Ulrich et al. 2006). These papers proposed identification of the uncertainty sources,starting from a clear declaration of the measured species, quantification of theseuncertainty sources, and, finally, a combination of these individual sources toestimate the standard and expanded uncertainty.

The present study presents the results obtained when applying a strategy forquantitative measurement uncertainty estimation of codeine in pharmaceuticalformulations using UV-VIS spectrophotometry based on an Ishikawa type diagram.

EXPERIMENTAL

Materials and Reagents

Codeine phosphate hemihydrate was purchased from Matthay MacFarlanSmith (lot 08-00883) and the pharmaceutical formulation containing 15mg=capletwas purchased from Ozone, Bucharest, Romania. All the reagents used in this studywere analytical grade. The NaOH used for solution preparation was obtained fromMerck (Germany). Ultra pure water was used for the preparation of the solutionsused in this study.

Apparatus

The spectrophotometric measurements were realized using a LAMDA 750UV-VIS-NIR apparatus (Perkin Elmer Life and Analytical Sciences).

Procedures

The calibration curve for codeine determination was obtained by using 11standard solutions in the range 1.18 � 10�4–9.06 � 10�4mol=L in 0.1M NaOHsolution. The absorbance of the solution was measured at k¼ 284 nm wavelength.

Figure 1. Structural formula of codeine phosphate hemyhidrate.

1208 I. DIACONU ET AL.

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

In the context of uncertainty evaluation, the specification of the measuredspecies demands a clear and ambiguity free declaration of what is measured, as wellas a quantitative expression that links the value of the measured species to theparameters on which it depends.

In order to list the uncertainty sources, it is very convenient to use thecause-effect diagram because it shows how the sources link to each other indicatingtheir influence on the result. This diagram also helps to avoid the doubling of theeffects.

The cause-effect diagram presents the main uncertainty sources present in thisstudy. The main branches represent the parameters that influence the result. Theseparameters are presented in Eq. (1):

Qsample ¼ c100 � V100 � 10�3=msample ð1Þ

where

Qsample – the analyte quantity, mol=kgc100 – the analyte concentration in the 100mL volumetric flask, mol=LV100 – the volume of the 100mL volumetric flask, mLmsample – the codeine sample mass taken, kg.

After identifying the uncertainty sources the next step is to quantify the uncertainty.The final step is calculating the expanded uncertainty, choosing carefully the cover-age factor.

Uncertainty Due to Concentration, c100

The codeine quantity is calculated from an established calibration curve. Thus,starting from a 9.85� 10�4mol=L stock solution, resulted from the weighting of0.4000 g of codeine and bringing it quantitatively to 1000mL volumetric flask withultra pure water, 11 standard solutions of codeine in 100mL volumetric flasks withconcentration ranging between 1.18� 10�4–9.06� 10�4mol=L using 1M NaOH sol-ution were prepared. All eleven solutions were measured three times. In this case, theuncertainty due to the standard solution was low enough to be neglected.

The uncertainty of the measured sample is given by Eq. (2):

uðcÞ ¼ srb

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1

nþ 1

pþ ðc� �ccÞ2

sxx

sð2Þ

where sr ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPn

j¼1½Yj�ðbxiþaÞ�n�2

r

sr – residual standard deviationn – number of measurements used for calibration curvep – number of measurements used to obtain the concentration of the samplec – analyte concentration in the unknown sample, mol=L�cc – average of standard solution, mol=L

UNCERTAINTY MEASUREMENT IN CODEINE TABLETS 1209

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

sxx ¼P

ðci � �ccÞ2Yj – analytical signal of the measurement jj – index for the number of measurements made in order to obtain the calibrationcurvei – index for the number of solution for the calibrationb – calibration curve slope, L=mola – calibration curve intercept

If the equation of the calibration curve has the form mentioned in Eq. (3):

y ¼ aþ bx ð3Þ

where:

y – analytical signal, absorbancea – calibration curve interceptb – calibration curve slope, L=molx – analyte concentration, mol=L,

the concentration c100 is obtained from the calibration curve equation.The sample solution was measured ten times (p¼ 10, the number of measure-

ments made for obtaining the concentration.

Uncertainty of the Discharge of 100mL Volumetric Flask

The uncertainty in case of the repeatability of the discharge of the 100mLvolumetric flask was determinate using an experiment consisting in filling up andweighting a 100mL volumetric flask with standard solution.

Uncertainty Associated with the Sample Mass msample

The sample mass was obtained through difference between the weighting glasswith and without the analyte.

RESULTS AND DISCUSSION

After the uncertainty sources were identified according to the cause-effectdiagram, these were evaluated and their magnitude was determined.

Uncertainty Due to Concentration, c100

After measuring the absorbance of the standard solution, the values for theabsorbance were obtained and presented in Table 1.

The calibration curve is described by Eq. (3), and the result of the linearregression is presented in Table 2.

The sample solution was measured ten times, thus obtaining the analyteconcentration c100. The results are presented in Table 3.

1210 I. DIACONU ET AL.

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

Realizing the average of the standard solution, we obtained the average of thevalues �cc¼ 5.12 � 10�4mol=L.

Knowing that the equation of the calibration curve is y¼ 1615.1��0.0028, theparameters of the regression curve were identified such as: slope b¼ 1615.1 L=moland the intercept a¼ –0.0028. For the determination of the calibration curve, elevensolutions were measured three times (total number of measurements is n¼ 33).

Thus:

sxx ¼ 6:83 � 10�7 ð4Þ

sr ¼ 8:29 � 10�2 ð5Þ

uðc100Þ ¼ 2:07 � 10�6 mol=L ð6Þ

In conclusion, the uncertainty for the sample was c100¼ 3.64 � 10�4mol=L whileu(c100)¼ 2.07 � 10�6mol=L, so the standard relative uncertainty was:

uðc100Þc100

¼ 2; 07 � 10�6

3:64 � 10�4¼ 5:68 � 10�3 ð7Þ

Table 1. Calibration data for codeine phosphate

i c mol �L�1 A1 A2 A3 A

1 1.18 � 10�4 0.193 0.194 0.193 0.193

2 1.97 � 10�4 0.315 0.314 0.314 0.314

3 2.76 � 10�4 0.443 0.444 0.444 0.444

4 3.55 � 10�4 0.570 0.570 0.570 0.570

5 4.33 � 10�4 0.693 0.692 0.693 0.693

6 5.12 � 10�4 0.811 0.811 0.811 0.811

7 5.91 � 10�4 0.960 0.961 0.960 0.960

8 6.70 � 10�4 1.085 1.085 1.085 1.085

9 7.49 � 10�4 1.206 1.205 1.206 1.206

10 8.28 � 10�4 1.335 1.334 1.334 1.334

11 9.06 � 10�4 1.461 1.461 1.461 1.461

Table 2. Calculated parameters of the linear regression analysis

Statistic parameter Value

Regression line equation y¼ 1615.1��0.0028

Slope of the regression line, b 1615.1

Standard deviation of the slope, sb 7.3323

Intersection to the origin, a �0.0028

Standard deviation of intersection, sa 0.0041

Correlation coefficient, R 0.9999

Determination coefficient, R2 0.9998

Relative standard deviation, RSD 0.0003

Number of freedom degrees, n 9

UNCERTAINTY MEASUREMENT IN CODEINE TABLETS 1211

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

Uncertainty of the Discharge of 100mL Volumetric Flask

The volume has three major influences: the calibration at the manufacturing ofthe volumetric flask, repeatability, and temperature.

The calibration at the manufacture of the volumetric flask (labeled onthe volumetric flask). The value of the deviation from the nominal volume for a100mL volumetric flask is indicated by the manufacturer as being �0.08mL (at20�C). Taking in account that the standard deviation is not given with a certain levelof confidence or with information regarding to the distribution, the value of thestandard uncertainty will be calculated assuming a triangular distribution. Thusthe uncertainty associated with the discharge of the 100mL volumetric flask dueto calibration u(V100_cal) is:

uðV100 calÞ ¼0:08ffiffiffi

6p ¼ 3:26 � 10�3 mL ð8Þ

Repeatability. After the filling and weighting experiment of a typical 100mLvolumetric flask, there was established a standard uncertainty of the volumetric flaskthat was established at 0.02mL, due to repeatability u(V100_rep).

Temperature. According to the manufacturer, the volumetric flask wascalibrated at a temperature of 20�C, while the temperature in the laboratory variedwith Dt¼�4�C. The uncertainty from this effect is calculated with the estimation ofthe temperature range and the volume dilatation coefficient. Because the dilatationof the volume of liquid is much larger than the dilatation of the volumetric flask,only the first one was taken into account. The volume dilatation coefficient, c, ofwater is 2.1 � 10�4�C�1. Thus starting from the aforementioned data, the uncertaintyfor the 100mL volumetric flask DV100 becomes:

DV100 ¼ V100 � c � Dt ð9Þ

Table 3. Experimental results for concentration c100

i Codeine found mtablet g Absorbance c mol �L�1

1 0.1550 0.590 3.63 � 10�4

2 0.1553 0.591 3.64 � 10�4

3 0.1552 0.592 3.64 � 10�4

4 0.1551 0.590 3.63 � 10�4

5 0.1550 0.590 3.63 � 10�4

6 0.1554 0.591 3.64 � 10�4

7 0.1551 0.592 3.64 � 10�4

8 0.1553 0.591 3.64 � 10�4

9 0.1553 0.590 3.63 � 10�4

10 0.1552 0.591 3.64 � 10�4

Average 0.1551 3.64 � 10�4

1212 I. DIACONU ET AL.

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

where:

DV100 – the uncertainty of the 100mL volumetric flaskV100 – the volume of the 100mL volumetric flaskc – volume dilatation coefficientDt – temperature variation in the laboratory

Thus, we obtain an uncertainty for the 100mL volumetric flask of 0.084mL.Assuming the temperature variation is a rectangular distribution the standard

uncertainty for the 100mL volumetric flask due to the temperature effect will beu(V100_temp):

uðV100 tempÞ ¼4 � 2; 1 � 10�4 � 100ffiffiffi

3p ¼ 0:048mL ð10Þ

Thus, the standard uncertainty due to the discharge of the 100mL volumetric flaskwill be:

uðV100Þ ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðuðV100 calÞÞ2 þ ðuðV100 repÞÞ2 þ ðuðV100 tempÞÞ2

qð11Þ

uðV100Þ ¼ 0:0038mL

The standard uncertainty will be:

uðV100ÞV100

¼ 3:8186 � 10�5 ð12Þ

Uncertainty Associated with the Sample Mass msample

At the quantification of the analyte mass we counted three uncertainty sources:sensitivity, linearity, and repeatability.

In order to assure the traceability of the results, the mass was expressed in kg,according to the International System of Units.

Sensitivity. Sensitivity can be neglected because the weighting is made by adifference on the same balance on the short range of the weighted mass.

Linearity. The data from the manufacturer indicated a linearity value of�0.0001 g. The evaluation of the uncertainty was made assuming a rectangular dis-tribution in order to convert the contribution to linearity into standard uncertainty.The contribution of linearity needed to be considered twice in the determination ofthe standard uncertainty (for tare and for the analyte mass):

u ¼ 0; 0001 � 10�3ffiffiffi3

p ¼ 5:77 � 10�8 kg ð13Þ

UNCERTAINTY MEASUREMENT IN CODEINE TABLETS 1213

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

Repeatability. From the calibration certificate of the balance, the uncertaintyassociated with repeatability is 0.0003 g.

In conclusion, the uncertainty due to sample mass u(msample) becomes:

uðmsampleÞ ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2xð5:77 � 10�8Þ2 þ ð0:0003 � 10�3Þ2

q¼ 3:1091 � 10�7 kg ð14Þ

The relative uncertainty due the sample mass is:

uðmsampleÞmsample

¼ 3:1091 � 10�7

0:15 � 10�3¼ 2:00 � 10�3 ð15Þ

The quantity of codeine in tablets, expressed as mol=kg, was calculated usingEq. (1). Thus, we obtain a quantity of 2.34 � 10�1mol=kg. The intermediate valuesand their standard uncertainties are presented in Table 4.

To calculate the composed uncertainty of the sample quantity Qsample, thestandard uncertainty is calculated according to (16):

uðQsampleÞQsample

¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiuðV100ÞV100

� �2

þ uðc100Þc100

� �2

þ uðmsampleÞmsample

� �2s

ð16Þ

Table 4. Summary of contribution to the measurement uncertainty for determination of codeine

phosphate from tablets through UV-Vis Spectrometry

Parameter Value

Standard

uncertainty, u(x)

Relative standard

uncertainty, u(x)=x

Volume, V100 (mL) 100 3.81 � 10�3 3.82 � 10�5

Sample concentration, c100 (mol �L�1) 3.64 � 10�4 2.07 � 10�6 5.68 � 10�3

Mass sample, msample (kg) 0.1551 � 10�3 3.10 � 10�7 2.00 � 10�3

Figure 2. Cause-effect diagram for the analysis of codeine from tablets.

1214 I. DIACONU ET AL.

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

Thus:

Qsample ¼ 2:34 � 10�1 mol=kg

uðQsampleÞ ¼ 1:41 � 10�3 mol=kg

Considering a confidence level of 95%, and a coverage factor k¼ 2, theexpanded uncertainty u(Qsample) is:

uðQsampleÞ ¼ 2 � 1:41 � 10�3 ¼ 2:82 � 10�3 mol=kg ð17Þ

The cause-effect diagram for the analysis of codeine tablets is shown in Fig. 2.The contribution of the different parameters and their influence to the uncertaintyare illustrated in Fig. 3.

CONCLUSIONS

The present study illustrates the realization of a cause-effect analysis in order toestimate the uncertainty in the measuring of codeine from pharmaceutical formula-tions through UV-VIS spectrometry. The estimation of the uncertainty componentsproved to be a good way for the experimental model to obtain low contributionto the uncertainty of the analytical result. In the present experiment, the majorcontribution to the uncertainty is represented by the contribution of the sampleconcentration. An insignificant contribution is represented by the contribution ofthe discharge of the 100mL volumetric flask. The result was found to be 2.34 � 10�1

� 2.82 � 10�3mol=kg.

REFERENCES

CITAC=EURACHEM. 2000.Quantifying uncertainty in analytical measurement. EURACHEM.

Figure 3. Illustration of contributions to uncertainty budget.

UNCERTAINTY MEASUREMENT IN CODEINE TABLETS 1215

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010

Dimitrovskat, A., S. Trajkovic-Jolevskat, A. Nancovskat, and M. Ilievska. 1995. Determi-nation of propyphenazone, paracetamol, caffeine and codeine phosphate with thin layerchromatography. Bull. Chem. Tech. Macedonia 1: 39–41.

European Pharmacopoeia. 2008. European Pharmacopoeia, 6th ed. Strasbourg, France:European Pharmacopoeia Council.

Hindson, B. J., P.S. Francis, S. D. Purcell, and N. W. Barnett. 2007. Determination of opiatealkaloids in process liquors using capillary electrophoresis. J. Pharma. Biomed. Anal. 43:1164–1168.

ISO. 1995. ISO guide to the expression of uncertainty in measurement, 1st ed. Switzerland: ISO.Lachenmeier, K., F. Musshoff, and B. Madea. 2006. Determination of opiates and cocaine in

hair using automated enzyme immunoassay screening methodologies followed by gaschromatographic–mass spectrometric (GC–MS) confirmation. Forensic Sci. Int. 159:189–199.

Papoutsis, I., P. Nikolaou, S. Athanaselis, C. Spiliopoulou, and C. Maravelias. 2008.Development and validation of a GC=EI-MS method for the determination of morphine,codeine and 6-acetyl-morphine in whole blood. Tox. Lett. 180S: S32–S246.

Pascual, J. A., and J. Sanagustın. Fully automated analytical method for codeine quantifi-cation in human plasma using on-line solid-phase extraction and high-performance liquidchromatography with ultraviolet detection. J.Chromatogr. B 2: 295–302.

Tanase, I., G. L. Radu, A. Pana, and M. Buleandra. 2006. Validarea Metodelor Analitice.Bucharest, Romania: Printech.

Ulrich, J. C., H. M. Shihomatsu, M. A. Hortellani, and J. H. S. Sarkis. 2006. Estimate ofUncertainty of Measurement in the Determination of Methylmercury in Fish Samples byFIA-CV-AAS. Rio de Janiero, Brazil: IMEKO XVIII World Congress.

World Health Organization (WHO) http://www.who.int. Accessed 3 September 2009.Zhang, Q.-L., J.-J. Xu, X.-Y. Li, H.-Z. Lian, and H-Y. Chen. 2007. Determination of

morphine and codeine in urine using poly(dimethylsiloxane) microchip electrophoresis withelectrochemical detection. J. Pharma. Biomed. Anal. 43: 237–242.

1216 I. DIACONU ET AL.

Downloaded By: [Bunaciu, Andrei] At: 19:00 18 May 2010