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Spectrochimica Acta Part B 54 1999.

827 833

Determination of trace elements in pharmaceutical substances by graphite furnace atomic absorption spectrometry and total reection X-ray uorescence after ow injection ion-exchange preconcentration
U Kelko-Levai, A. I. Varga, K. Zih-Perenyi, A. Lasztity Department of Inorganic and Analytical Chemistry, L. Eot os Uni ersity, P.O. Box 32, Budapest 112, H-1518 Hungary Received 5 October 1998; accepted 9 February 1999

Abstract Flow injection iminodiacetic acid ethyl cellulose IDAEC. microcolumn preconcentration and graphite furnace atomic absorption spectrometry determination of trace metals Cd, Co, Ni, Pb. were carried out without decomposition of the drug matrix. The two forms of chromium CrIII. and CrVI. were separated using IDAEC and anion exchanger diethylaminoethyl DE.-cellulose, respectively. The detection limits of trace elements in pharmaceutical substances sugars, sorbitol, mannitol, paracetamol, amidopyrine, chloral hydrate. after a 10-fold preconcentration in 1 5% mrv solution of pharmaceuticals were in the 0.3 29 ng gy1 range. The measured concentration of trace elements in substances investigated was below 100 ng gy1. The spike recovery was close to 100%. The capability of total reection X-ray uorescence technique for the determination of trace elements in pharmaceuticals with and without preconcentation was explored. 1999 Elsevier Science B.V. All rights reserved.
Keywords: ETAAS; TXRF; Flow injection; Preconcentration; Impurities; Drugs; Speciation; Cr

This paper was published in the Special Issue of the Third European Furnace Symposium, Prague, Czech Republic, June 1998. Corresponding author. Fax: q36-1-209-0602.

0584-8547r99r$ - see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S 0 5 8 4 - 8 5 4 7 9 9 . 0 0 0 3 6 - 1

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Kelko-Le A. ai et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 54 (1999) 827 833

1. Introduction In the quality control of drugs and medicines, determination of inorganic impurities is important. Some of these trace elements are toxic, some of them even in ultratrace concentrations could decrease the stability of active ingredients. Some of the impurities could be indicators of non-adequate handling and storage or be used as ngerprints for the source of drug crude material. Heavy metal tests are included in all pharmacopoeias. They are non-specic tests for metals based on the formation of coloured sulphide species. These tests are not universally applicable to check for all potential heavy metals catalysts: Pd, Pt, Rh; or impurities from stainless steel vessels: Cr, Fe, Ni, etc.. and the used reagents H 2 S and thioacetamide are toxic. Because the sensitivity and selectivity of these tests in most cases is not sufcient for the recent industrial requirements, the determination of trace inorganic impurities by instrumental methods has become more important for the quality control of pharmaceutical substances. For some substances, e.g. sugars, the test limit for lead - 0.5 g gy1 . is specied in the European Pharmacopoeia w1x. Prior to analysis by graphite furnace atomic absorption spectrometry GFAAS. the sugar sample has to be decomposed in a polyuorocarbonate bomb for 5 h at 150C because a direct analysis is difcult due to high content of organic matter. In compounds synthesized with Pd catalysts, Pd is also determined by GFAAS w2x, inductively coupled plasma-mass spectrometry ICP-MS. and axial inductively coupled plasma-atomic emission spectroscopy ICPAES. techniques w3,4x. Lewen et al. w5x examined ICP-MS and axial ICP-AES as an alternative to the USP heavy metal test. Total-reection X-ray uorescence spectrometry TXRF. is a multi-element method with low detection limits, small sample mass requirement and simple quantication in a wide dynamic range w6x. Its rst application in drug analysis was for the discrimination between batches of the same pharmaceutical product originating from different production or purication processes by their element nger-

prints measured by TXRF directly or after digestion of samples w7x. The matrix of pharmaceuticals has a big variety because of their different chemical composition. In many cases direct measurement of trace elements without decomposition of the matrix could not be accomplished either by GFAAS or TXRF. Coupling a preconcentration step to GFAAS and TXRF could reduce the matrix effect, improve the sensitivity, and could avoid the tedious and time-consuming sample decomposition of substances. A separation allows also to distinguish between anionic and cationic forms of metals and thus to investigate possible interactions between the drug matrix and metals. Iminodiacetic acid ethyl cellulose IDAEC. chelating ion exchanger has ideal properties to be used in ow injection FI. on-line systems as microcolumn lling for multi-element preconcentration of trace metals w8x. Stability constants of metal IDAEC chelates were determined and used to predict the sorption of ultratrace elements from highly mineralised medicinal waters on a FI microcolumn for preconcentration prior to their spectrochemical determination w9x. Chromium-III and CrVI. contents in rain water samples were determined using IDAEC and diethylaminoethyl DE.-cellulose for preconcentration w10x. The aims of this work were: 1. to study the possibility of using a FI-IDAEC microcolumn preconcentration procedure for separation of trace metals from drug matrices prior to their GFAAS and TXRF measurement; 2. to develop a screening metal test using direct TXRF method or coupling it with FI-microcolumn preconcentration; and 3. to determine CrIII. and CrVI. levels in drugs by preconcentration on IDAEC and DE-cellulose microcolumns.

2. Experimental 2.1. Reagents Throughout the study, high-purity water dis-

Kelko-Le A. ai et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 54 (1999) 827 833

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tilled water puried on ion-exchange celluloses . was used. The ammonium acetate buffer solution was prepared by isotherm distillation. All reagents were of analytical grade. AAS metal standards 1.000 g ly1 supplied by Merck Darmstadt, Germany. were used for preparation of calibration solutions. IDAEC was synthesised from aminoethyl cellulose w11x. The copper capacity of the exchanger was 0.8 mmol gy1 . The capacity of DE-cellulose Whatman. was 1.0 mmol gy1 . The pharmaceutical substances analysed are sugars sucrose, glucose, lactose., sorbitol, mannitol, amidopyrine 4-dimethylamino-1,5-dimethyl2-phenyl-4-pyrazolin-3-one., chloral hydrate and indomethacin. 2.2. Instrumentation

Atomika EXTRA IIA Germany. spectrometer. Operating conditions were as follows: Excitation: line-focused X-ray tubes, Be window; Mo anode excitation energy: 17.5 keV, 200 m Mo cut-off lter.; W anode excitation energy: 35 keV, 300 m cut-off Ni lter.. Mo or W tube operated at 50 kV and 25 mA. Incident angle: 6. Detection: SiLi. detector; 2000 channel analyser, resolution: 0.16 keV at 6 keV.. Measuring time: 600 s. Analytical lines in keV.: Cr K 5.41; Fe K 6.40; Co K 6.93; Ni K 7.47; Cu K 8.04; Zn K 8.63; Pb L 10.55 Mo anode.; Cd K 23.11 W anode.. The samples were spiked with 200 ng mly1 Sr as internal standard and dried 10 20- l aliquots. on pre-cleaned quartz glass carriers. 2.3. Preconcentration

A Perkin-Elmer Model 3110 atomic absorption spectrometer with deuterium background corrector, a Perkin-Elmer HGA-600 graphite furnace with pyrolytic graphite coated tubes and Lvov platforms, and a Perkin-Elmer AS-60 autosampler were used. All measurements were run with 20 l sampling volumes using gas stops and integrated absorbance readings during the atomization step. Operating conditions and analytical parameters are listed in Table 1. TXRF measurements were performed on
Table 1 Measuring conditions Temperature program Drying C. Ramprhold time s. Drying C. Ramprhold time s. Pyrolysis C. Ramprhold time s. Cooling C. Ramprhold time s. Atomisation C. Ramprhold time s. Gas ow at atomisation Modier
a

Preconcentration of Cd, Co, CrIII., Ni and Pb was performed by on-line ow injection FI. on a microcolumn lled with 40 mg of IDAEC in NH 4-form. The drug samples were dissolved in water, in case of paracetamol in a 20% vrv methanol water mixture to get a 1 5% mrv solution. A dual four-way valve assembly was applied for preconcentration and elution of trace elements w9x. The eluent was 2 mol ly1 HNO3 at ow rate 2.0 ml miny1 . The eluates were col-

Cd 228.8 nm 110 10r10 180 10r20 500 1r30 20 1r15 1500 0r5 0

Co 241.0 nm 110 10r10 180 10r20 1400 1r30 20 1r15 2500 0r5 0

Cr 357.9 nm 110 10r10 130 1r35 1650 1r30 20 1r15 2500 0r5 Gas stop 0.05 mg MgNO3 .2a

Ni 232.0 nm 110 10r10 180 10r20 1400 1r30 20 1r15 2500 0r5 0

Pb 283.3 nm 110 10r10 180 10r20 700 1r30 20 1r15 1900 0r5 0

5 ml of 1% mrv solution.

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Kelko-Le A. ai et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 54 (1999) 827 833
Table 2 Analysis results of NIST 1643c by FI-GFAAS IDAEC microcolumn preconcentration Element Certied g ly1 . 12.2 " 1.0 23.5 " 0.8 19.0 " 0.6 60.6 " 7.3 35.3 " 0.9 Founda,b g ly 1 . 11.4 " 0.6 24.0 " 1.1 18.1 " 0.8 58.2 " 1.9 34.9 " 1.4

lected into vessels of the AS-60 autosampler. The sample volume loaded was 10.0 ml, the eluted volume was 0.95 1.00 ml. For CrVI. 30 mg of DE-cellulose in Cl-form was used as exchanger. The eluent was 1 mol ly1 HCl. Four calibration solutions before enrichment were prepared within the concentration ranges: Cd, CrIII., CrVI.: 0 0.2; Co: 0 4.0; Pb: 0 5.0; and Ni: 0 10.0 g ly1 . For direct TXRF measurements, a 1% mrv solution of indomethacin in a mixture of 98% acetone 2% HCl 1 mol ly1 . was prepared. The metal spike additions were: Cd, Co, CrIII., Cu, Pb: 100 ng mly1 ; Ni: 200 ng mly1 . The eluates were spiked with 200 ng mly1 Sr as the internal standard and measured by TXRF. 3. Results and discussion 3.1. FI-IDAEC microcolumn-GFAAS determination of trace metals in pharmaceutical substances The optimal pH for preconcentration on IDAEC is 5.0 w9x. At that pH, calibration graphs were constructed by preconcentration of four calibration solutions. The sorption ratio of total metal in the eluate and in the loaded solution. was close to 100% Cd: 100 " 3.4; Co: 95 " 2.8; CrIII.: 83 " 2.9; CrVI.: 94 " 3.4; Ni: 91 " 3.9; Pb: 94 " 2.6.. Addition of 20% methanol to water

Cd Co CrIII. Ni Pb
a b

The sample was 20 times diluted for Cd, 200 times.. Average of ve measurements.

had no effect on the sorption. Good linearity of the calibration graphs with regression coefcients better than 0.997 proves that in the concentration range used for calibration the differences in sorption are within the experimental error. The preconcentration procedure was tested by analysis of NIST 1643c Trace Elements in Water Standard Reference Material. Appropriate agreement was obtained for all the elements as seen in Table 2. Ten millilitres of the solution of pharmaceuticals between 1% and 5% mrv was loaded on IDAEC microcolumn and after elution the trace metal content was measured by GFAAS. The content was below the quantication limit 5 = DL. except for cadmium where 2.8, 2.7 and 2.0 ng gy1 Cd were found in chloral hydrate, amidopyrine and paracetamol, respectively.

Table 3 Spike recovery on IDAEC microcolumn from pharmaceutical substances by FI-GFAAS Substances Cd Added Glucose Lactose Sucrose Sorbitol Mannitol Amidopyrine Paracetamol Chloral hydrate 4.0 4.0 4.0 2.0 2.0 20 10 20
y1

Co Found 3.6 4.1 3.6 1.9 1.6 20.2a 9.9b 16a Added 80 80 80 80 80 400 400 400 Found 80.8 76.8 78.4 80.8 84 404a 420a 392a

CrIII. Added 20 20 20 20 20 20 20 Found 20.6 19.8 19.8 20.6 21.6 14.8 22.0

CrVI. Added 20 20 20 20 20 Found 19.6 18.4 16.8 19.8 21.8

Ni Added 200 200 200 200 200 100 500 100 Found 208 194 204 190 192 97 495b 99

Pb Added 100 100 100 100 100 500 500 Found 102 90 99 101 98 520a 500a

Note. Results in ng g ; sample concentrations: 5% mrv. a Sample concentration: 1%. b Sample concentration: 2%.

Kelko-Le A. ai et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 54 (1999) 827 833
Table 4 Precision and accuracy of spike recoveries from sorbitol and mannitol by FI-GFAAS Element Spike ng gy1 . Sorbitol Precision R.S.D. %. 3.2 5.3 4.9 3.8 9.1 5.7 Recovery %. 93 101 97 100 96 101 Mannitol Precision R.S.D. %. 8.1 5.2 5.2 9.5 4.5 Recovery %. 80 100 99 96 99

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Cd Co CrIII. CrVI. Ni Pb

2.0 40.0 20.0 20.0 100.0 50.0

Note. Seven samples were preconcentrated.

The procedure was, therefore, checked by spike recoveries and the results are given in Table 3. For most of the elements and substances the recovery was between 90 and 100%. Increasing the amidopyrine concentration from 1% to 5% mrv the recovery of Pb and Cd decreased to 60% and 69%, respectively. However, the 74% recovery of CrIII. in 5% solution allows the analysis by standard addition. Precision of the procedure was estimated using spiked 5% mrv solutions of sorbitol and mannitol. Results of seven separate analyses including preconcentration are in Table 4. The analytical recoveries were close to 100% and the R.S.D. was approximately 5%, only in the case of Ni was the R.S.D. higher. Detection limits based on the 3 criterion were calculated from seven preconcentrated blanks. The detection limits ng gy1 . depend on the
Table 5 Detection limits in ng gy1 by FI-GFAAS Substance Cd Co CrIII. CrVI.

amount of the substance used for preconcentration. The heavy metal limits expressed in lead as specied for individual drugs in the European Pharmacopoeia w1x are one or two orders of magnitude higher than the detection limits of the method see Table 5.. It may be expected that introducing new spectrochemical methods ICPAES and ICP-MS. to drug quality control existing limits will be replaced by much lower and element-specic ones. Based on a usage of AAS for some substances lower limit values have already been specied in European Pharmacopoeia for lead 0.5 g gy1 . and nickel 1.0 g gy1 .. 3.2. Total-reexion X-ray uorescence analysis The possibility to use TXRF as an alternative to the Heavy Metal Test in the European Pharmacopoeia was studied. To improve the sensitivity

Ni

Pb

Limit values w1x Pb Ni Heavy metals

Glucose Sucrose Lactose Mannitol Sorbitol Chloral hydrate Amidopyrine Paracetamol

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.7b

2.3 2.3 2.3 2.3 2.3 11.5a 11.5a 11.5a

4.8 4.8 4.8 4.8 4.8 4.8 4.8

4.3 4.3 4.3 4.3 4.3

7.2 7.2 7.2 7.2 7.2 7.2 7.2 18.0b

5.8 5.8 5.8 5.8 5.8 5.8 29.0a 29.0a

500 500 5000 500 500 10 000 1000 1000 1000

20 000

Note. Loading 10 ml 5% mrv solution. a 1% solution. b 2% solution.

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Kelko-Le A. ai et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 54 (1999) 827 833

Table 6 TXRF determination of metals after IDAEC-microcolumn preconcentration Element Preconcentration blank Preconcentration blank q spike 200 g ly1 . 182 213 224 205 226 208 S.D. n s 3. 18.0 17.1 19.4 18.3 7.2 8.3 10 times preconcentrated 20 g ly 1 . 202 218 182 220 232 224 S.D. n s 3. 17.0 12.4 14.9 6.1 3.1 6.7

Cd Co Cu Fe Ni Pb

Not detected Not detected - 20 - 30 - 30 - 20 Note. Results in g ly1 .

of TXRF method and to avoid the sample digestion step the same preconcentration procedure as for GFAAS was used. To test the effect of the eluate matrix on the TXRF measurement results from a preconcentration blank spiked with 200 g ly1 metal solutions were compared with the 10-fold preconcentrated 20 g ly1 metal solutions Table 6.. The results show that the sorption was above 90% for most of the elements investigated. The standard deviations were calculated from three separate measurements. The substances analysed by GFAAS and TXRF are the same. Other elements preconcentrated on IDAEC Cu, Fe, Zn, etc.. can be determined in the same way. The found level of impurities for most of the elements was lower than 0.5 g gy1 . The recovery of spike additions of 80 1000 ng gy1 after preconcentration on the IDAEC microcolumn was also checked. The concentration of

spikes in individual drugs is usually one order of magnitude lower than the specied heavy metal limits Table 5.. The recoveries presented in Table 7 are better than 70%, though with high uncertainty due to high background. To get more accurate results, larger additions, longer measuring time or a higher preconcentration factor should be used. For substances not soluble in water or in water methanol ethanol. mixtures a direct TXRF method can be used. As an example, the trace metal level in indomethacin was determined using a 1% mrv solution of the drug in acetone. The recoveries of 100 ng mly1 spikes of all the elements see Table 8. are acceptable considering that the limit value of the Heavy Metal Test is 10 g gy1 . Detection limits of elements in the eluate as derived from spectral background noise when us-

Table 7 Spike recovery of elements on IDAEC microcolumn from pharmaceutical substances by TXRF Substances CdII. Added Glucose Lactose Sucrose Sorbitol Mannitol Amidopyrinea Paracetamola Chloral hydratea 100 100 20 20 20 Found 82 72 - 25 - 27 - 19 CoII. Added 80 80 80 400 400 400 Found - 90 - 95 100 440 395 350 NiII. Added 200 200 200 100 200 1000 1000 1000 Found 212 167 180 132 210 770 955 760 PbII. Added 100 100 100 100 100 500 500 Found 74 122 94 127 137 472 370

Note. Results in ng gy1 ; concentration of samples: 5% mrv. a Concentration: 1% mrv.

Kelko-Le A. ai et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 54 (1999) 827 833
Table 8 Spike recovery from 1% mrv solution of indomethacin by direct TXRF Metals 1% indomethacin found ng mly1 . - 20 - 20 - 20 - 25 - 30 1% indomethacin qspike 100 ng mly 1 . found ng mly1 . 107 86 104 96 126

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Acknowledgements

Co Cr Cu Ni Pb

We would like to thank Toth Karolyne for her technical assistance. The research was sponsored by the National Foundation of Scientic Research OTKA No. A196r95r450..

References
w1x European Pharmacopoeia, 3rd ed., Council of Europe, Strasbourg, 1997. Levai, w2x A. A. Lasztity, K. Zih-Perenyi, Zs. Horvath, Microchem. J. 58 1998. 272 280. w3x N. Lewen, M. Schenkenberger, T. Larkin, S. Conder, H.G. Brittain, J. Pharm. Biomed. Anal. 13 1995. 879 883. w4x M. Schenkenberger, N. Lewen, 1998 Winter conference on plasma spectrochemistry, Scottsdale, Arizona, Poster PF 24, ICP Information Newsletter, Special Ed. 23 1998. 333. w5x N. Lewen, S. Mathew, M. Schenkenberger, 1998 Winter conference on plasma spectrochemistry, Scottsdale, Arizona, Poster PF 23, ICP Information Newsletter, Special Ed., 23 1998. 333. w6x R. Klockenkamper, A. Bohlen, J. Anal. At. Spectrom. 7 1992. 273 279. w7x M. Wagner, P. Rostam-Khani, A. Wittershagen, C. Rittmeyer, B.O. Kolbesen, H. Hoffman, Spectrochim. Acta Part B 52 1997. 961 965. w8x S. Caroli, A. Alimonti, F. Petrucci, Zs. Horvath, Anal. Chim. Acta 248 1991. 241 249. Levai, w9x Zs. Horvath, K. Zih-Perenyi, A. Mi A. Lasztity, crochem. J. 54 1996. 391 401. w10x Zs. Horvath, I. Varga, E. Meszaros, A. A. Lasztity, Molnar, Talanta 41 1994. 1165 1168. w11x Zs. Remport-Horvath, Gy. Nagydiosi, J. Inorg. Nucl. Chem. 37 1975. 767 769.

Note. Indomethacin was dissolved in a mixture of acetone and 1 mol ly1 HCl 98 q 2.; 200 ng mly1 Sr internal standard.

ing samples of only 20 l of eluate and 600 s measuring times during TXRF analysis were found to be at 10 g ly1 level. 4. Conclusions A FI-IDAEC microcolumn preconcentration has been coupled to GFAAS for the determination of heavy metals in sugars and drugs. This procedure separates, and enriches trace metals from pharmaceutical substances without decomposition of the matrix thus improving sensitivity and eliminating matrix effects in GFAAS determinations. Quantitation at ng gy1 levels is possible. Using different cellulose ion-exchangers for the preconcentration the ionic state of chromium in pharmaceutical products can be veried and quantied. The multielement TXRF method with or without preconcentration can provide an element specic screening procedure for heavy metals as well as for all other potential elements and inorganic impurities.

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