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Use of pralidoxime-Pd(II) complex for the spectrophotometric determination of the cholinesterase reactivator (PAM-2Cl) in the urine
Clinica Chimica Acru, 193 (1990) 119-124 Elsevier
119
CCA 04866
Use of pralidoxime-Pd( II) complex for the spectrophotometric determination of the cholinesterase reactivator (PAM-2Cl) in the urine Katarina
D. Karljiko~~-Raji~
and Branislava
S. StankoviC
Faculty of Ph~macy, rnstitu~e of Analytica/ ~herni~t~, Belgrade ~Yugo~lavia) (Received 15 April 1989; revision received 7 February 1990; accepted 30 August 1990) Key woruk Pralidoxime chloride: Antidote; Spectrophotometric Pralidoxime-Pd(II) complex; Urine
determination:
A simple and rapid method for the spectrophotometric determination of pralidoxime chloride in urine, based on complex formation with palladium(I1) without preliminary ~ner~~ation of the sample, is described. The proposed method is shown to be reproducible and in good augment with a reference method, which involved spectrophotomet~~ determination of co~esponding oximate ions in ammonium hydroxide solution at 336 nm. Our results indicate that the proposed method is reliable, rapid and of sufficient sensitivity for pralidoxime chloride analysis in urine.
Introduction Various pyridinium aldoximes are effective antidotes in organophosphorus poisoning, and are accepted as part of the therapeutic regimen against poisoning by anticholinesterase compounds. Pralidoxime (PAM) has undergone extensive clinical evaluation and is reported to be an effective antidote in humans [l-4]. Sp~trophotomet~c methods have been used for meas~~ment of PAM in biological materials (urine, serum and blood). Several of these are based on the formation of the corresponding oximate ion followed by spectrophotometry at 335 nm [5,6] and this methodology has been subjected to mechanization [7]. Burger et al. [8] proposed the procedure for spectrophotometric determination of pralidoxime chloride in the urine by using sodium amminepentacyanoferrate (II) as color-forming reagent.
Correspondence to: K.D. KarljikovibRajiC, Bdgrade, Yugoslavia. ~9-8981/~/$03.50
Faculty of Pharmacy. Institute of Analytical Chemistry,
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
120
In our previous work we reported the use of ming reagent in determination of pralidoxime tions and tablets [9]. We now report on the spectrophotometric determination of PAM-2Cl
palladium(I1) chloride as colour-forchloride (PAM-2Cl) in water soluapplication of this principle to the in urine.
Materials and metho+ Reagents and solutions
Pralidoxime chloride (1-methyl-2-hydroxyiminomethyl pyridinium chloride (PAM-2Cl)) PAM-2Cl (purity > 99.5%) was ‘synthesized at the Laboratory of Organic Chemistry, Bosnalijek Company, Sarajevo. A freshly prepared aqueous solution of PAM-2Cl (5.7931 X lop4 mol/l) was used as the stock standard solution; it was stable for several days. Palladium(I1) chloride solution (10F2 mol/l) in hydrochloric acid was prepared as reported [9]. B&ton-Robinson’s buffer solution [lo] was prepared by mixing 100 ml of an aqueous solution of acetic, boric and phosphoric acids (each 0.08 mol/l) with 47.3 ml of sodium hydroxide solution (0.4 mol/l) to adjust the pH to 6.45.4 85 ml of 2 mol/l potassium chloride solution was added to adjust the ionic strength to 0.2 mol/l. The ionic strength (CL)of the final solution for the proposed spectrophotometric determination with Pd(I1) was kept constant at 0.3 mol/l by addition of 2 mol/l potassium chloride solution. Trichloroacetic acid solution was 0.6 mol/l and ammonium hydroxide was 13.31 mol/l. All chemicals were of analytical-grade (Merck). Double-distilled water was used throughout. Random urine samples were obtained from ten healthy persons 3 males and 7 females (aged 23-55 yr). Preparation of the calibration curve for the proposed method (Method A)
Potassium chloride solution (0.50 ml) and palladium(I1) chloride solution (0.20 ml) were placed in a 5 ml standard flask and a portion (0.02-0.40 ml) of PAM-2Cl standard solution was added. The pH was then adjusted to 6.45 by adding 2.50 ml of Britton-Robinson’s buffer and the solution was diluted to 5.00 ml with water. After mixing, the absorbance at 327 nm was measured after 10 mm against a reagent blank. All measurements were made at room temperature (25 + 0.5 o C). Preparation of the calibration curve for the comparison method (Method B)
Portions (0.02-0.32 ml) of PAM-2Cl standard solution were diluted to volumes of 1.00 ml with water and 1.00 ml of the trichloroacetic acid was added. After mixing 1.00 ml of the mixture was transferred to a cell, 1.30 ml of water and 0.20 ml of the ammonium hydroxide solution were added. The reactants were mixed and the absorbance at 336 nm measured immediately against a reagent blank. All measurements were made at room temperature (25 f 0.5 o C).
121
Determination of PAM-2CI in the urine The urine was diluted 1 : 10 with water and 0.20 ml of the diluted urine was used in both methods. In method A, the diluted urine was added to the mixture of potassium chloride and palladium(H) chloride solutions, and the portion (0.05; 0.10 or 0.20 ml) of PAM-2Cl standard solution that correspond to the concentrations (1.448; 2.896 or 5.793 mmol/l of native urine) was added and the procedure was followed as described. A reagent blank, against which the absorbance of the sample solutions were measured, contained the corresponding 0.20 ml of the urine sample but without PAM-2Cl. In method B, to the diluted urine sample, same aliquot (0.05; 0.10 or 0.20 ml) of PAM-2Cl solution was added diluted to 1.00 ml with water mixed with 1.00 ml of trichloroacetic acid, centrifuged if necessary and the procedure was followed as described. Also in this method a reagent blank contained corresponding 0.20 ml of same urine sample but without PAM-2Cl. Statistics The data were statistically evaluated using the significance tests: comparison an experimental mean with a known value and Student’s t test [ll].
of
Results
Our preliminary experiments, of the absorbance measurements at 327 nm of pralidoxime-Pd(I1) complex from the urine sample, prove that the complex formation is completed within 10 min, and therefore all further measurements were done after that time. Calibration curves for both determinations were prepared as described in assay procedure without the addition of the urine. Three replicate analysis were performed for each calibration curve. Using the proposed method, PAM-2Cl can be determined in concentration range 0.579-11.586 mmol/l and with the reference method between 0.579-9.264 mmol/l of native urine (Fig. 1). To determine the imprecision of the proposed and the reference methods, urine samples containing three different PAM-2Cl concentrations were measured (Table I). The calculated values of 1t 1 (0.098-1.903), for both methods and for all concentrations are less than the critical value 2.262 (P’ = 0.05; 0 = 9). There was no evidence of systematic error. Comparison data for PAM-2Cl determination for three different concentrations in PAM-2Cl-spiked urine, as assayed by the proposed and the reference method, yielded the following linear regression equation: y = -1.469
x
1O-2 + 0.987x; r = 0.9999; S,,, = 0.0157;
t, = 0.761 < t,,,, ( x-axis, reference method; y-axis, proposed method).
122
Fig. 1. Calibration graphs: Method A (curve 2): y = 2.25 X 10s3 + 4.38 X IO-2~; r = 0.9999; S,, x = 4.22 X 10e4. Method B (curve 2); y = 4.08 X 10W3t 7.71 X IO-%; r = 0.9998; & = 4.76 x fOL3.
The detection limit (defined as the analyte ~ncentration giving the signal equal to the blank signal, ya, plus three standard deviation of the blank, S,) for the proposed method is 0.0288 pmol/ml and for the reference method 0.1854 ~mol/ml of PAM-2Cl of native urine. Analytical recovery experiments on pralidoxime-spiked urine (n = 10) are shown in Table II. Recovery ranged from 99.17-99.86% (mean 99.39%) across a wide PAM-2CI concentration range.
TABLE I Precision data for the determination of pralidoxime-spiked Added found
1.448 mmol/l
(0-18:
2.896 mmol/i
5.793 mmol/i
(?I =lO)
A (n =lO)
B (n =lO)
A
(?I =lO)
(n =lO)
B (n =lO)
1.446 0.0644 0.0204 4.45
1.471 0.0598 0.0189 4.07
2.873 0.0687 0.0217 2.39
2.938 0.0698 0.0221 2.37
5.754 0.0671 0.0212 1.16
5.839 0.0889 0.0281 1.52
Ab
x SDC SR CV(W Calculated c values Tabulated f value
mine samples a
-Bb
0.853
1.991
2.289
2.093
2.093
2.093
P=O.O5)
a 0.2 ml diluted urine (I : 10). b A, proposed method; B, reference method. ’ Inter-assay SD.
123 TABLE II Analytical recovery data (Method A) of pr~do~me
added to urine
PAM-2Cl added (mmol/l)
PAM-Xl measured @mow
1.448 2.896 4.345 5.793 7.241
1.446 2.873 4.318 5.754 7.181
Recovery @) 99.86 99.21 99.37 99.34 99.17 Mean 99.39 + 0.27
r = 0.9999; y = 9.39 x 10 - 3 +0.99Osx; $_ = 7.01 x 10-3; t, =I277 < tO.OS = 3.182 (0= 3).
Discussion Due to the increased use of pyridinium oximes there is an increased interest to find out new analytical methods for their determination in pharmaceutical preparations as well as in the biological media. The determination of pyridinium oximes in the blood, serum and urine is of a great practical importance both in the experimental and in the clinical medicine. For the successful therapy with these antidotes, it is of a special importance to follow their level in the blood, as well as the speed of their elimination in the urine. In most studies the oximes were administered by the intravenous and intramuscular route. The oral is less remended because these drugs, being charged compounds, are poorly absorbed from the gastro-intestinal tract 112,131. After intravenous administration of PAM-2C1, about 86% of a dose is excreted in the urine in 24 h, mostly within the first 3 h. Pralidoxime mesylate is more readily absorbed from the gastro-intestinal tract, and after its oral administration about 30% of a dose is excreted in the 24-h urine [14,15]. We report the new spectrophotometric procedure for determination of PAM-2Cl in the urine based on the yellow complex formation with Pd(II), without preliminary mineralization of the sample. The optimum experimental conditions for pralidoxime-Pd(II) complex formation (opt~um pH, reagent con~ntration, ionic strength, standing time) have been studied previously [9]. Since we found that this method was suitable for accurate, sensitive and reproducible determination of PAM-2Cl both in pure and in drug, we tried to apply it for PAM-2Cl determination in the urine. The conditions in the proposed method comparing, to those of the reference method [6], which is concerned with the absorption of the corresponding oximate ion of basic PAM-2Cl solution, are more suitable because degradation process of oxime does not occur which is the main disadvantage in the reference method. The Student’s t test gave no significant difference between the means obtained by the proposed and the reference method, except for the highest measured concentration (Table I). The results obtained by the reference method are slightly higher, so the difference between means is more pronounced at higher concentration
124
that could be the reason why significant difference was obtained for 5.793 mmol/l of PAM-2CI. In the both methods absorbances of pralidoxime-spiked urine samples were measured against the corresponding reagent blanks which contained the same urine sample but without PAM-2Cl. This was necessary, because in the proposed method Pd(I1) could react with same other nitrogen compounds from the urine, and in the reference method the different urine samples without PAM-2Cl had different absorbance at 336 m-n. For determination of PAM-2Cl in clinical experiments, control urine sample is taken from the patient before treatment with the drug. The obtained results showed that the proposed method satisfy the r~uirements for determination of drugs in biological material. Acknowledgement The authors are grateful to the Serbian Republic Research Fund for the financial support. References 1 Quinby GE. Further therapeutic experience with pralidoximes in organic phosphorus poisoning. J Am Med Assoc 1%~187:202-206. 2 Barkman R, Edgren B, Sundwall A. Self-a~~stration of pralidoxime in nerve gas poisoning with a note on the stability of the drug. J Pharm Pharmacol 1963;XV(lO):671-677. 3 Sidell FR, Groff WA, Elfin RI. Blood levels of oxime and symptoms in humans after single and multiple doses of 2-pyridine aldoxime methochloride. J Pharfn Sci 1%9;58:1093-1098. 4 Quinby GE. Feasibility of prophylaxis by oral pralidoxime. Arch Environ Health 1968;16:812-820. 5 Creasey NH, Green AL. 2-hydroxyiminomethyl-N-methylpyridinium methanesulphonate (P2S), an antidote to organophosphorus poisoning. Its preparation, estimation and stability. J Pharm Pharmaco1 1959:11:485-490. 6 MaksimoviC M, Vojvodib V. Selection of the method for determination of oxime content in biological material. (in Serbocroation, with summary in English) Arh Hig Rada Toksikol 1969;20:173-176. 7 Groff WA, EBin RI. A new and rapid determination of pyridinium aldoximes in blood and urine. Chn Chem 1969;15:72-83. 8 Burger N, Smerih Z, Hankonyi V. Use of ~ntacy~ofe~a~ (II) complexes as &or-forming reagents in dete~ations of obidoxime in the blood serum and pr~do~me in the urine. Acta Pharm Yugosl 1986;36:15-18. 9 Karljikovi&Raji& K, Stankovi& B, Granov A, Binenfeld Z. Use of p~a~um(II) chloride as colourforming reagent in determination of pralidoxime chloride in water and tablets. J Pharm Biomed Anal 1987;5(2):773-780. 10 Coeh-Frugoni JA. Tampone universale di B&ton e Robinson a form ionica constante. Gazz Chim Ital 1957;87:403-407. 11 Miller JC, Miller JN. Statistics for analytical chemistry, 2nd ed. UK; Ellis Horwood Ltd., 1988;53:101. 12 Side11 FR, Groff WA, Elfin RI. Blond levels of oxime and symptoms in humans after single and multiple doses of 2-pyridine aldoxime methochloride. J Pharm Sci 1969;58:1093-1098. 13 MaksimoviC M. Oximes HI-6 and PAM-2Cl: comparative pharmacokinetic studies after intramuscular and oral administration to the rat (in Serbocroation, with summary in English). Arh Hig Rada Toksikol 1979;30:227-239. 14 Jossolson J, Sidefl FR. Effect of intravenous thiamin on pralidoxime kinetics. Chn Pharmacof Ther 1978;2ql):95-1~. 15 Clark& Isolation and Id~tifi~tion of Drugs, 2 ed. London: The Pharmaceutical Press, 1986;915.
119
CCA 04866
Use of pralidoxime-Pd( II) complex for the spectrophotometric determination of the cholinesterase reactivator (PAM-2Cl) in the urine Katarina
D. Karljiko~~-Raji~
and Branislava
S. StankoviC
Faculty of Ph~macy, rnstitu~e of Analytica/ ~herni~t~, Belgrade ~Yugo~lavia) (Received 15 April 1989; revision received 7 February 1990; accepted 30 August 1990) Key woruk Pralidoxime chloride: Antidote; Spectrophotometric Pralidoxime-Pd(II) complex; Urine
determination:
A simple and rapid method for the spectrophotometric determination of pralidoxime chloride in urine, based on complex formation with palladium(I1) without preliminary ~ner~~ation of the sample, is described. The proposed method is shown to be reproducible and in good augment with a reference method, which involved spectrophotomet~~ determination of co~esponding oximate ions in ammonium hydroxide solution at 336 nm. Our results indicate that the proposed method is reliable, rapid and of sufficient sensitivity for pralidoxime chloride analysis in urine.
Introduction Various pyridinium aldoximes are effective antidotes in organophosphorus poisoning, and are accepted as part of the therapeutic regimen against poisoning by anticholinesterase compounds. Pralidoxime (PAM) has undergone extensive clinical evaluation and is reported to be an effective antidote in humans [l-4]. Sp~trophotomet~c methods have been used for meas~~ment of PAM in biological materials (urine, serum and blood). Several of these are based on the formation of the corresponding oximate ion followed by spectrophotometry at 335 nm [5,6] and this methodology has been subjected to mechanization [7]. Burger et al. [8] proposed the procedure for spectrophotometric determination of pralidoxime chloride in the urine by using sodium amminepentacyanoferrate (II) as color-forming reagent.
Correspondence to: K.D. KarljikovibRajiC, Bdgrade, Yugoslavia. ~9-8981/~/$03.50
Faculty of Pharmacy. Institute of Analytical Chemistry,
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
120
In our previous work we reported the use of ming reagent in determination of pralidoxime tions and tablets [9]. We now report on the spectrophotometric determination of PAM-2Cl
palladium(I1) chloride as colour-forchloride (PAM-2Cl) in water soluapplication of this principle to the in urine.
Materials and metho+ Reagents and solutions
Pralidoxime chloride (1-methyl-2-hydroxyiminomethyl pyridinium chloride (PAM-2Cl)) PAM-2Cl (purity > 99.5%) was ‘synthesized at the Laboratory of Organic Chemistry, Bosnalijek Company, Sarajevo. A freshly prepared aqueous solution of PAM-2Cl (5.7931 X lop4 mol/l) was used as the stock standard solution; it was stable for several days. Palladium(I1) chloride solution (10F2 mol/l) in hydrochloric acid was prepared as reported [9]. B&ton-Robinson’s buffer solution [lo] was prepared by mixing 100 ml of an aqueous solution of acetic, boric and phosphoric acids (each 0.08 mol/l) with 47.3 ml of sodium hydroxide solution (0.4 mol/l) to adjust the pH to 6.45.4 85 ml of 2 mol/l potassium chloride solution was added to adjust the ionic strength to 0.2 mol/l. The ionic strength (CL)of the final solution for the proposed spectrophotometric determination with Pd(I1) was kept constant at 0.3 mol/l by addition of 2 mol/l potassium chloride solution. Trichloroacetic acid solution was 0.6 mol/l and ammonium hydroxide was 13.31 mol/l. All chemicals were of analytical-grade (Merck). Double-distilled water was used throughout. Random urine samples were obtained from ten healthy persons 3 males and 7 females (aged 23-55 yr). Preparation of the calibration curve for the proposed method (Method A)
Potassium chloride solution (0.50 ml) and palladium(I1) chloride solution (0.20 ml) were placed in a 5 ml standard flask and a portion (0.02-0.40 ml) of PAM-2Cl standard solution was added. The pH was then adjusted to 6.45 by adding 2.50 ml of Britton-Robinson’s buffer and the solution was diluted to 5.00 ml with water. After mixing, the absorbance at 327 nm was measured after 10 mm against a reagent blank. All measurements were made at room temperature (25 + 0.5 o C). Preparation of the calibration curve for the comparison method (Method B)
Portions (0.02-0.32 ml) of PAM-2Cl standard solution were diluted to volumes of 1.00 ml with water and 1.00 ml of the trichloroacetic acid was added. After mixing 1.00 ml of the mixture was transferred to a cell, 1.30 ml of water and 0.20 ml of the ammonium hydroxide solution were added. The reactants were mixed and the absorbance at 336 nm measured immediately against a reagent blank. All measurements were made at room temperature (25 f 0.5 o C).
121
Determination of PAM-2CI in the urine The urine was diluted 1 : 10 with water and 0.20 ml of the diluted urine was used in both methods. In method A, the diluted urine was added to the mixture of potassium chloride and palladium(H) chloride solutions, and the portion (0.05; 0.10 or 0.20 ml) of PAM-2Cl standard solution that correspond to the concentrations (1.448; 2.896 or 5.793 mmol/l of native urine) was added and the procedure was followed as described. A reagent blank, against which the absorbance of the sample solutions were measured, contained the corresponding 0.20 ml of the urine sample but without PAM-2Cl. In method B, to the diluted urine sample, same aliquot (0.05; 0.10 or 0.20 ml) of PAM-2Cl solution was added diluted to 1.00 ml with water mixed with 1.00 ml of trichloroacetic acid, centrifuged if necessary and the procedure was followed as described. Also in this method a reagent blank contained corresponding 0.20 ml of same urine sample but without PAM-2Cl. Statistics The data were statistically evaluated using the significance tests: comparison an experimental mean with a known value and Student’s t test [ll].
of
Results
Our preliminary experiments, of the absorbance measurements at 327 nm of pralidoxime-Pd(I1) complex from the urine sample, prove that the complex formation is completed within 10 min, and therefore all further measurements were done after that time. Calibration curves for both determinations were prepared as described in assay procedure without the addition of the urine. Three replicate analysis were performed for each calibration curve. Using the proposed method, PAM-2Cl can be determined in concentration range 0.579-11.586 mmol/l and with the reference method between 0.579-9.264 mmol/l of native urine (Fig. 1). To determine the imprecision of the proposed and the reference methods, urine samples containing three different PAM-2Cl concentrations were measured (Table I). The calculated values of 1t 1 (0.098-1.903), for both methods and for all concentrations are less than the critical value 2.262 (P’ = 0.05; 0 = 9). There was no evidence of systematic error. Comparison data for PAM-2Cl determination for three different concentrations in PAM-2Cl-spiked urine, as assayed by the proposed and the reference method, yielded the following linear regression equation: y = -1.469
x
1O-2 + 0.987x; r = 0.9999; S,,, = 0.0157;
t, = 0.761 < t,,,, ( x-axis, reference method; y-axis, proposed method).
122
Fig. 1. Calibration graphs: Method A (curve 2): y = 2.25 X 10s3 + 4.38 X IO-2~; r = 0.9999; S,, x = 4.22 X 10e4. Method B (curve 2); y = 4.08 X 10W3t 7.71 X IO-%; r = 0.9998; & = 4.76 x fOL3.
The detection limit (defined as the analyte ~ncentration giving the signal equal to the blank signal, ya, plus three standard deviation of the blank, S,) for the proposed method is 0.0288 pmol/ml and for the reference method 0.1854 ~mol/ml of PAM-2Cl of native urine. Analytical recovery experiments on pralidoxime-spiked urine (n = 10) are shown in Table II. Recovery ranged from 99.17-99.86% (mean 99.39%) across a wide PAM-2CI concentration range.
TABLE I Precision data for the determination of pralidoxime-spiked Added found
1.448 mmol/l
(0-18:
2.896 mmol/i
5.793 mmol/i
(?I =lO)
A (n =lO)
B (n =lO)
A
(?I =lO)
(n =lO)
B (n =lO)
1.446 0.0644 0.0204 4.45
1.471 0.0598 0.0189 4.07
2.873 0.0687 0.0217 2.39
2.938 0.0698 0.0221 2.37
5.754 0.0671 0.0212 1.16
5.839 0.0889 0.0281 1.52
Ab
x SDC SR CV(W Calculated c values Tabulated f value
mine samples a
-Bb
0.853
1.991
2.289
2.093
2.093
2.093
P=O.O5)
a 0.2 ml diluted urine (I : 10). b A, proposed method; B, reference method. ’ Inter-assay SD.
123 TABLE II Analytical recovery data (Method A) of pr~do~me
added to urine
PAM-2Cl added (mmol/l)
PAM-Xl measured @mow
1.448 2.896 4.345 5.793 7.241
1.446 2.873 4.318 5.754 7.181
Recovery @) 99.86 99.21 99.37 99.34 99.17 Mean 99.39 + 0.27
r = 0.9999; y = 9.39 x 10 - 3 +0.99Osx; $_ = 7.01 x 10-3; t, =I277 < tO.OS = 3.182 (0= 3).
Discussion Due to the increased use of pyridinium oximes there is an increased interest to find out new analytical methods for their determination in pharmaceutical preparations as well as in the biological media. The determination of pyridinium oximes in the blood, serum and urine is of a great practical importance both in the experimental and in the clinical medicine. For the successful therapy with these antidotes, it is of a special importance to follow their level in the blood, as well as the speed of their elimination in the urine. In most studies the oximes were administered by the intravenous and intramuscular route. The oral is less remended because these drugs, being charged compounds, are poorly absorbed from the gastro-intestinal tract 112,131. After intravenous administration of PAM-2C1, about 86% of a dose is excreted in the urine in 24 h, mostly within the first 3 h. Pralidoxime mesylate is more readily absorbed from the gastro-intestinal tract, and after its oral administration about 30% of a dose is excreted in the 24-h urine [14,15]. We report the new spectrophotometric procedure for determination of PAM-2Cl in the urine based on the yellow complex formation with Pd(II), without preliminary mineralization of the sample. The optimum experimental conditions for pralidoxime-Pd(II) complex formation (opt~um pH, reagent con~ntration, ionic strength, standing time) have been studied previously [9]. Since we found that this method was suitable for accurate, sensitive and reproducible determination of PAM-2Cl both in pure and in drug, we tried to apply it for PAM-2Cl determination in the urine. The conditions in the proposed method comparing, to those of the reference method [6], which is concerned with the absorption of the corresponding oximate ion of basic PAM-2Cl solution, are more suitable because degradation process of oxime does not occur which is the main disadvantage in the reference method. The Student’s t test gave no significant difference between the means obtained by the proposed and the reference method, except for the highest measured concentration (Table I). The results obtained by the reference method are slightly higher, so the difference between means is more pronounced at higher concentration
124
that could be the reason why significant difference was obtained for 5.793 mmol/l of PAM-2CI. In the both methods absorbances of pralidoxime-spiked urine samples were measured against the corresponding reagent blanks which contained the same urine sample but without PAM-2Cl. This was necessary, because in the proposed method Pd(I1) could react with same other nitrogen compounds from the urine, and in the reference method the different urine samples without PAM-2Cl had different absorbance at 336 m-n. For determination of PAM-2Cl in clinical experiments, control urine sample is taken from the patient before treatment with the drug. The obtained results showed that the proposed method satisfy the r~uirements for determination of drugs in biological material. Acknowledgement The authors are grateful to the Serbian Republic Research Fund for the financial support. References 1 Quinby GE. Further therapeutic experience with pralidoximes in organic phosphorus poisoning. J Am Med Assoc 1%~187:202-206. 2 Barkman R, Edgren B, Sundwall A. Self-a~~stration of pralidoxime in nerve gas poisoning with a note on the stability of the drug. J Pharm Pharmacol 1963;XV(lO):671-677. 3 Sidell FR, Groff WA, Elfin RI. Blood levels of oxime and symptoms in humans after single and multiple doses of 2-pyridine aldoxime methochloride. J Pharfn Sci 1%9;58:1093-1098. 4 Quinby GE. Feasibility of prophylaxis by oral pralidoxime. Arch Environ Health 1968;16:812-820. 5 Creasey NH, Green AL. 2-hydroxyiminomethyl-N-methylpyridinium methanesulphonate (P2S), an antidote to organophosphorus poisoning. Its preparation, estimation and stability. J Pharm Pharmaco1 1959:11:485-490. 6 MaksimoviC M, Vojvodib V. Selection of the method for determination of oxime content in biological material. (in Serbocroation, with summary in English) Arh Hig Rada Toksikol 1969;20:173-176. 7 Groff WA, EBin RI. A new and rapid determination of pyridinium aldoximes in blood and urine. Chn Chem 1969;15:72-83. 8 Burger N, Smerih Z, Hankonyi V. Use of ~ntacy~ofe~a~ (II) complexes as &or-forming reagents in dete~ations of obidoxime in the blood serum and pr~do~me in the urine. Acta Pharm Yugosl 1986;36:15-18. 9 Karljikovi&Raji& K, Stankovi& B, Granov A, Binenfeld Z. Use of p~a~um(II) chloride as colourforming reagent in determination of pralidoxime chloride in water and tablets. J Pharm Biomed Anal 1987;5(2):773-780. 10 Coeh-Frugoni JA. Tampone universale di B&ton e Robinson a form ionica constante. Gazz Chim Ital 1957;87:403-407. 11 Miller JC, Miller JN. Statistics for analytical chemistry, 2nd ed. UK; Ellis Horwood Ltd., 1988;53:101. 12 Side11 FR, Groff WA, Elfin RI. Blond levels of oxime and symptoms in humans after single and multiple doses of 2-pyridine aldoxime methochloride. J Pharm Sci 1969;58:1093-1098. 13 MaksimoviC M. Oximes HI-6 and PAM-2Cl: comparative pharmacokinetic studies after intramuscular and oral administration to the rat (in Serbocroation, with summary in English). Arh Hig Rada Toksikol 1979;30:227-239. 14 Jossolson J, Sidefl FR. Effect of intravenous thiamin on pralidoxime kinetics. Chn Pharmacof Ther 1978;2ql):95-1~. 15 Clark& Isolation and Id~tifi~tion of Drugs, 2 ed. London: The Pharmaceutical Press, 1986;915.