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Liquid Chromatographic Determination of Propoxyphene and Norpropoxyphene in Plasma and Breast Milk
Liquid Chromatographic Determination of Propoxyphene and Norpropoxyphene in Plasma and Breast Milk ROBERT L.
KUNKA*",
CHAN-LOI YONG**,
CAROLE
F. LADIK',
AND
THEODORE R. BATES*"
Received October 21, 1983,from the, Department of Pharmaceutics, School of Pharmacy, University of PittsburP h 1Pittsburgh, PA 15261 and §Magee Womens Hospital, Pittsburgh, PA 15213. Accepted for publication May 3, 1984. Present addresses: Marion Laboratories, Kansas City, Mo 641 37. ' Searle Pharmaceuticals, Chicago, IL 60680. A Texas Southern University, Houston, Texas, 77035. '
0 A sensitive and specific high-performance liquid chromatographic (HPLC) procedure was developed for determination of propoxyphene and norpropoxyphene in plasma and breast milk. The compounds were isolated from the biological specimen by extraction, the organic phase was evaporated to dryness, and the residue was redissolved in mobile phase [acetonitrile:0.002 M H2S04(1 :l)].The resultant solution was then injected into an HPLC system utilizing a C,8 reversed-phase analytical column and a variable-wavelength detector set at 205 nm. Under these conditions the method has a sensitivity of 20 ng/mL using 1 mL of plasma or milk. The within-run coefficient of variation for both compounds varied between 6.2 and 8.9%within the concentration range tested. Applicability of the method was demonstrated in a nursing mother who received multiple oral doses of propoxyphene. Abstract
propoxyphene in tablet and capsule dosage forms. This report describes a sensitive and specific HPLC method capable of determining propoxyphene and norpropoxyphene in small volumes of plasma and breast milk.
Experimental Section
Propoxyphene HCl (4-dimethylamino-1,2-diphenyl-3methyl-2-propionoxybutanehydrochloride; 1) and norpropoxyphene maleate (4-methylamino-1,2-dipheny1-3-methyl-Z-propionoxybutane maleate; 2 ) were supplied by Eli Lilly and Company. 2-Diethylaminoethyl-2,2-diphenylvalerateHCl, 3, (SKF 525-A, Smith, Kline and French), acetonitrile, (HPLC grade, Burdick and Jackson Laboratories, Muskegon, MI), and Fisher Scientific Company HPLC-grade chloroform, n-butyl Propoxyphene (1) is a widely prescribed oral analgesic. In chloride, ether (purified), and reagent grade alcohol were used recent years it has been responsible for a number of poisonings as received. and Upon multiple dosing, norpropoxyphene ( 2 ) ,the The M-6000A solvent delivery HPLC system (Waters Assoactive metabolite of the drug, accumulates in the plasma. ciates) was equipped with WISP automatic sample injector and Therefore, any analysis of propoxyphene should also be capable a SF770 spectroflow monitor variable-wavelength UV-absorbof determining norpropoxyphene. ance detector (Schoeffel Instruments, Westwood, NJ) operated A number of assays are available for determining propoxyat 205 nm. phene and norpropoxyphene in biological fluids. Most of these A reversed-phase p-Bondapack C1, chromatographic column methods utilize GC separation with flame-ionization detec(30 cm x 3.9 mm i.d., Waters Associates) was used for separat i ~ n . ~ -These '' methods have two common shortcomings: large tion and was operated at room temperature. The mobile phase volumes of biological sample are required (2-10 mL plasma), was acetonitrile:0.002 M H,SO, (1:l)at a flow rate of 1.5 mL/ and on-column thermolytic degradation of drug may O C C U ~ . ' ~ ~ ' ' ~ min. The detector sensitivity was set at 0.02 AUFS and the Two high-performance liquid chromatographic (HPLC) assays chart speed was 30 cm/h. have been but these assays can only determine Stock solutions of propoxyphene HCl and norpropoxyphene maleate were made in alcohol. Appropriate volumes of these solutions were diluted to make five spiking solutions containing both compounds in concentrations of 40-480 ng of free base/ 50 pL. A separate alcoholic solution containing 600 ng/50 pL of 3 was prepared and used as the internal standard. The extraction procedure was a modificztion of that reported C H ~ORJ by Verebely and Inturrisi." All extractions were performed in I I silanized glassware (to prevent drug adsorption) at high speed R'R'NCH-C -C-CH2 for 15 min on an Eberbach reciprocating shaker (VWR ScienI I tific Co.), and phase separations were effected by centrifugation at 20OOxg for 5 min. Plasma or breast milk (0.2-1.0 mL), adjusted to 1.0 mL with blank biological fluid when necessary, was spiked with 50 pL of the internal standard solution. After addition of 0.5 mL of 1.0 M carb0nate:bicarbonate buffer (pH 9.80), propoxyphene and norpropoxyphene were coextracted into 10.0 mL of n-butyl 1, R' = R2 = CH3, R3 = COCHzCH3 chloride and then re-extracted from the organic (upper) phase 2, R' = H, R2 = CH3, R3 = COCHZCH3 into 5.0 mL of 0.2 M HCl. Following centrifugation, the organic 2a, R' = CH3, R2 = COCHzCH3,R3 = H phase was removed by aspiration and the aqueous phase was washed with 5.0 mL of ether. The washed aqueous phase was CsHs then alkalinized to -pH 12 with 0.5 mL of 4 M NaOH (to I convert norpropoxyphene to its amide form, 2a), and propoxCH~(CHZ)~CCOZCHZCHZN(CZH~)Z yphene and the amide of norpropoxyphene were coextracted I into 10.0 mL of chloroform. The aqueous (upper) phase was CsHs removed by aspiration, and the organic phase was transferred to a clean 15-mL centrifuge tube and evaporated to dryness 3 0022-3549/85/0100-0103$0l.OO/O 0 1985, American Pharmaceutical Association
Journal of Pharmaceutical Sciences 1 103 Vol. 74, No. 1, January 1985
under nitrogen at 55°C. The residue was then redissolved in 200 pL of mobile phase, and 100 pL of this solution was injected into the HPLC column. Linear calibration plots of peak height ratio of propoxyphene or the amide of norpropoxyphene to internal standard versus drug or metabolite concentration were constructed daily by analyzing spiked plasma or breast milk samples (40-480 ng/ mL) by the procedure described.
Results and Discussion The detection wavelengths chosen for the proposed HPLC method were based on the UV absorption characteristics of propoxyphene in the mobile phase. A spectrophotometric scan (Beckman Model 25 Spectrophotomer) indicated that propoxyphene does not absorb well a t the wavelengths commonly used in the HPLC determinations of other drugs (i.e., 254 and 280 nm). However, the absorption of UV.energy by propoxyphene is substantial in the low UV range. A wavelength of 205 nm was used for detection and produced chromatograms devoid of endogenous interferences in the sample due to the extensive cleanup used. The UV response was more than adequate to detect propoxyphene and norpropoxyphene in patient plasma and breast milk samples. Figure 1 shows typical chromatograms obtained following extraction of blank plasma, spiked plasma, and plasma of a nursing mother who received multiple oral doses of propoxyphene. The HPLC method is specific as indicated by the separate peaks for the three compounds of interest and by virtue of selective extractions. Propoxyphene, the amide of norpropoxyphene, and the internal standard were eluted at 5.7, 7.6, and 8.8 min, respectively. The initial peaks in each chromatogram are due to endogenous substances present in the biological specimens. Chromatograms similar to those shown in Fig. 1 were obtained from extracted breast milk samples. The peak height ratios of propoxyphene or the amide of norpropoxyphene to internal standard were linear with concentration over the range of 40-480 ng of propoxyphene or norpropoxyphene/mL of plasma or breast milk. The mean (SD) linear regression equations for five calibration curves assayed over three months were y = 0.00634 (0.00073)~- 0.088 (0.050) with a correlation coefficient (SD) of 0.991 (0.006) for propoxyphene, and y = 0.00608 (0.00175)~- 0.003 (0.125) with a correlation coefficient (SD) of 0.987 (0.009) for norpropoxyphene. Detection of propoxyphene and norpropoxyphene at 205 nm was sensitive to 20 ng/mL of biological fluid. The mean overall recoveries of propoxyphene and norpropoxyphene (+SD) from plasma (n = 12) and breast milk (n = 12) were 81.8% _t 5.9% and 85.5 k 6.2%, respectively. The
MINUTES
Figure 1-Chromatograms of propoxyphene (A), the amide of norpropoxyphene (B),and internal standard (C) in blank plasma (left panel); spiked plasma, 320 ng/mL of propoxyphene and norpropoxyphene (middle panel); and subject plasma, 108 nglmL of propoxyphene and 372 nglmL of norpropoxyphene (right panel). 104
Journal of Pharmaceutical Sciences Vol. 74, No. 1, January 1985
1
0.00
2.00
4.00
6.00
8.00
10.00
Hours Figure 2-Concentration-time profile for propoxyphene in plasma (C) and breast milk (D), and norpropoxyphene in plasma (A) and breast milk (6)in a nursing mother following multiple-dose oral adminstration of 65mg propoxyphene.
within-day (intra-assay) coefficients of variation of the HPLC method in spiked plasma and breast milk (n = 8) at 40, 160, and 480 ng/mL were 8.9, 6.2, and 6.9% for propoxyphene and 8.6, 7.4, and 6.8% for norpropoxyphene. The utility of the proposed HPLC method was demonstrated by determining propoxyphene and norpropoxyphene concentrations in nursing mothers following multiple dose oral administration of two 65-mg propoxyphene HCl capsules (Darvon) followed by one capsule every 4 h for a total of six capsules.17 The time courses of propoxyphene and norpropoxyphene in plasma and breast milk following the last dose in one subject are shown in Fig. 2. The HPLC method possesses the specificity and sensitivity required to monitor propoxyphene and norpropoxyphene concentrations in humans.
References and Notes 1. Finkle, B. S.; McCloskey, K. L.; Kiplinger, G. F.; Bennett, 1. F. J. Forensic Sci. 1 9 7 6 , 2 1 , 706. 2. Gustafson, A.; Gustafson, B. Acta Med. Scand. 1 9 7 6 , 2 0 0 , 241. 3. Sturner, W. Q.; Garriott, J. C. J. Am. Med. Assoc. 1973,223,1125. 4. Frinps. C. S.: Foster. L. B. Am. J. Clin. Pathol. 1970.53.944. 5. Chri;tensen,'H. Act; Pharmacol. Tozicol. 1977, 40, 289. ' 6. Nash, J. F.; Bennett, I. F.; Bopp, R. J.; Brunson, M. K.; Sullivan, H. R. J. Pharm. Sci. 1975,64,429. 7. Walle, T.; Ehrsson, H. Acta Pharm. Succica 1971, 8 , 27. 8. Cleemann, M. J. Chromatogr. 1977, 132,287. 9. Angelo, H. R.; Christensen, J. M. J. Chromatogr. 1977, 140, 280. 10. Manno, J.; Jain, N.; Crim, D.; Forney, R. J . Forensic Sci. 1970, 15, 403. 11. Wolen, R. L.; Gruber, C. M., Jr. Anal. Chem. 1 9 6 8 , 4 0 , 1243. 12. Verebely, K.; Inturrisi, C. E. J. Chromatogr. 1973, 75, 195. 13. Millard, B. J.; Sheinin, E. B.; Benson, W. R. J. Pharm. Sci. 1980, 69. 1177. 14. Sparacino, C. M.; Pellizzari, E. D.; Cook, C. E.; Wall, M. W. J . Chromatogr. 1973, 77, 413. 15. Gilpin, R. K.; Korpi, J. A.; Janicki, C. A. J . Chromatogr. 1975, -707 - . , -115 - -. 16. Borkan, S.; Wainer, I. W. J . Chromatogr. 1982, 240, 547. 17. Kunka, R. L.; Venkataramanan, R.; Stern, R. M.; Ladik, C. F. Clin. Pharmacol. Ther. 1 9 8 4 , 3 5 , 675.
Acknowledgments Supported in part by a grant from the Magee Womens Hospital Research Fund, Pittsburgh, PA. The authors thank Ms. Judy Cherevka for manuscript preparation and Ms. Rhonda Harrison for technical assistance.
KUNKA*",
CHAN-LOI YONG**,
CAROLE
F. LADIK',
AND
THEODORE R. BATES*"
Received October 21, 1983,from the, Department of Pharmaceutics, School of Pharmacy, University of PittsburP h 1Pittsburgh, PA 15261 and §Magee Womens Hospital, Pittsburgh, PA 15213. Accepted for publication May 3, 1984. Present addresses: Marion Laboratories, Kansas City, Mo 641 37. ' Searle Pharmaceuticals, Chicago, IL 60680. A Texas Southern University, Houston, Texas, 77035. '
0 A sensitive and specific high-performance liquid chromatographic (HPLC) procedure was developed for determination of propoxyphene and norpropoxyphene in plasma and breast milk. The compounds were isolated from the biological specimen by extraction, the organic phase was evaporated to dryness, and the residue was redissolved in mobile phase [acetonitrile:0.002 M H2S04(1 :l)].The resultant solution was then injected into an HPLC system utilizing a C,8 reversed-phase analytical column and a variable-wavelength detector set at 205 nm. Under these conditions the method has a sensitivity of 20 ng/mL using 1 mL of plasma or milk. The within-run coefficient of variation for both compounds varied between 6.2 and 8.9%within the concentration range tested. Applicability of the method was demonstrated in a nursing mother who received multiple oral doses of propoxyphene. Abstract
propoxyphene in tablet and capsule dosage forms. This report describes a sensitive and specific HPLC method capable of determining propoxyphene and norpropoxyphene in small volumes of plasma and breast milk.
Experimental Section
Propoxyphene HCl (4-dimethylamino-1,2-diphenyl-3methyl-2-propionoxybutanehydrochloride; 1) and norpropoxyphene maleate (4-methylamino-1,2-dipheny1-3-methyl-Z-propionoxybutane maleate; 2 ) were supplied by Eli Lilly and Company. 2-Diethylaminoethyl-2,2-diphenylvalerateHCl, 3, (SKF 525-A, Smith, Kline and French), acetonitrile, (HPLC grade, Burdick and Jackson Laboratories, Muskegon, MI), and Fisher Scientific Company HPLC-grade chloroform, n-butyl Propoxyphene (1) is a widely prescribed oral analgesic. In chloride, ether (purified), and reagent grade alcohol were used recent years it has been responsible for a number of poisonings as received. and Upon multiple dosing, norpropoxyphene ( 2 ) ,the The M-6000A solvent delivery HPLC system (Waters Assoactive metabolite of the drug, accumulates in the plasma. ciates) was equipped with WISP automatic sample injector and Therefore, any analysis of propoxyphene should also be capable a SF770 spectroflow monitor variable-wavelength UV-absorbof determining norpropoxyphene. ance detector (Schoeffel Instruments, Westwood, NJ) operated A number of assays are available for determining propoxyat 205 nm. phene and norpropoxyphene in biological fluids. Most of these A reversed-phase p-Bondapack C1, chromatographic column methods utilize GC separation with flame-ionization detec(30 cm x 3.9 mm i.d., Waters Associates) was used for separat i ~ n . ~ -These '' methods have two common shortcomings: large tion and was operated at room temperature. The mobile phase volumes of biological sample are required (2-10 mL plasma), was acetonitrile:0.002 M H,SO, (1:l)at a flow rate of 1.5 mL/ and on-column thermolytic degradation of drug may O C C U ~ . ' ~ ~ ' ' ~ min. The detector sensitivity was set at 0.02 AUFS and the Two high-performance liquid chromatographic (HPLC) assays chart speed was 30 cm/h. have been but these assays can only determine Stock solutions of propoxyphene HCl and norpropoxyphene maleate were made in alcohol. Appropriate volumes of these solutions were diluted to make five spiking solutions containing both compounds in concentrations of 40-480 ng of free base/ 50 pL. A separate alcoholic solution containing 600 ng/50 pL of 3 was prepared and used as the internal standard. The extraction procedure was a modificztion of that reported C H ~ORJ by Verebely and Inturrisi." All extractions were performed in I I silanized glassware (to prevent drug adsorption) at high speed R'R'NCH-C -C-CH2 for 15 min on an Eberbach reciprocating shaker (VWR ScienI I tific Co.), and phase separations were effected by centrifugation at 20OOxg for 5 min. Plasma or breast milk (0.2-1.0 mL), adjusted to 1.0 mL with blank biological fluid when necessary, was spiked with 50 pL of the internal standard solution. After addition of 0.5 mL of 1.0 M carb0nate:bicarbonate buffer (pH 9.80), propoxyphene and norpropoxyphene were coextracted into 10.0 mL of n-butyl 1, R' = R2 = CH3, R3 = COCHzCH3 chloride and then re-extracted from the organic (upper) phase 2, R' = H, R2 = CH3, R3 = COCHZCH3 into 5.0 mL of 0.2 M HCl. Following centrifugation, the organic 2a, R' = CH3, R2 = COCHzCH3,R3 = H phase was removed by aspiration and the aqueous phase was washed with 5.0 mL of ether. The washed aqueous phase was CsHs then alkalinized to -pH 12 with 0.5 mL of 4 M NaOH (to I convert norpropoxyphene to its amide form, 2a), and propoxCH~(CHZ)~CCOZCHZCHZN(CZH~)Z yphene and the amide of norpropoxyphene were coextracted I into 10.0 mL of chloroform. The aqueous (upper) phase was CsHs removed by aspiration, and the organic phase was transferred to a clean 15-mL centrifuge tube and evaporated to dryness 3 0022-3549/85/0100-0103$0l.OO/O 0 1985, American Pharmaceutical Association
Journal of Pharmaceutical Sciences 1 103 Vol. 74, No. 1, January 1985
under nitrogen at 55°C. The residue was then redissolved in 200 pL of mobile phase, and 100 pL of this solution was injected into the HPLC column. Linear calibration plots of peak height ratio of propoxyphene or the amide of norpropoxyphene to internal standard versus drug or metabolite concentration were constructed daily by analyzing spiked plasma or breast milk samples (40-480 ng/ mL) by the procedure described.
Results and Discussion The detection wavelengths chosen for the proposed HPLC method were based on the UV absorption characteristics of propoxyphene in the mobile phase. A spectrophotometric scan (Beckman Model 25 Spectrophotomer) indicated that propoxyphene does not absorb well a t the wavelengths commonly used in the HPLC determinations of other drugs (i.e., 254 and 280 nm). However, the absorption of UV.energy by propoxyphene is substantial in the low UV range. A wavelength of 205 nm was used for detection and produced chromatograms devoid of endogenous interferences in the sample due to the extensive cleanup used. The UV response was more than adequate to detect propoxyphene and norpropoxyphene in patient plasma and breast milk samples. Figure 1 shows typical chromatograms obtained following extraction of blank plasma, spiked plasma, and plasma of a nursing mother who received multiple oral doses of propoxyphene. The HPLC method is specific as indicated by the separate peaks for the three compounds of interest and by virtue of selective extractions. Propoxyphene, the amide of norpropoxyphene, and the internal standard were eluted at 5.7, 7.6, and 8.8 min, respectively. The initial peaks in each chromatogram are due to endogenous substances present in the biological specimens. Chromatograms similar to those shown in Fig. 1 were obtained from extracted breast milk samples. The peak height ratios of propoxyphene or the amide of norpropoxyphene to internal standard were linear with concentration over the range of 40-480 ng of propoxyphene or norpropoxyphene/mL of plasma or breast milk. The mean (SD) linear regression equations for five calibration curves assayed over three months were y = 0.00634 (0.00073)~- 0.088 (0.050) with a correlation coefficient (SD) of 0.991 (0.006) for propoxyphene, and y = 0.00608 (0.00175)~- 0.003 (0.125) with a correlation coefficient (SD) of 0.987 (0.009) for norpropoxyphene. Detection of propoxyphene and norpropoxyphene at 205 nm was sensitive to 20 ng/mL of biological fluid. The mean overall recoveries of propoxyphene and norpropoxyphene (+SD) from plasma (n = 12) and breast milk (n = 12) were 81.8% _t 5.9% and 85.5 k 6.2%, respectively. The
MINUTES
Figure 1-Chromatograms of propoxyphene (A), the amide of norpropoxyphene (B),and internal standard (C) in blank plasma (left panel); spiked plasma, 320 ng/mL of propoxyphene and norpropoxyphene (middle panel); and subject plasma, 108 nglmL of propoxyphene and 372 nglmL of norpropoxyphene (right panel). 104
Journal of Pharmaceutical Sciences Vol. 74, No. 1, January 1985
1
0.00
2.00
4.00
6.00
8.00
10.00
Hours Figure 2-Concentration-time profile for propoxyphene in plasma (C) and breast milk (D), and norpropoxyphene in plasma (A) and breast milk (6)in a nursing mother following multiple-dose oral adminstration of 65mg propoxyphene.
within-day (intra-assay) coefficients of variation of the HPLC method in spiked plasma and breast milk (n = 8) at 40, 160, and 480 ng/mL were 8.9, 6.2, and 6.9% for propoxyphene and 8.6, 7.4, and 6.8% for norpropoxyphene. The utility of the proposed HPLC method was demonstrated by determining propoxyphene and norpropoxyphene concentrations in nursing mothers following multiple dose oral administration of two 65-mg propoxyphene HCl capsules (Darvon) followed by one capsule every 4 h for a total of six capsules.17 The time courses of propoxyphene and norpropoxyphene in plasma and breast milk following the last dose in one subject are shown in Fig. 2. The HPLC method possesses the specificity and sensitivity required to monitor propoxyphene and norpropoxyphene concentrations in humans.
References and Notes 1. Finkle, B. S.; McCloskey, K. L.; Kiplinger, G. F.; Bennett, 1. F. J. Forensic Sci. 1 9 7 6 , 2 1 , 706. 2. Gustafson, A.; Gustafson, B. Acta Med. Scand. 1 9 7 6 , 2 0 0 , 241. 3. Sturner, W. Q.; Garriott, J. C. J. Am. Med. Assoc. 1973,223,1125. 4. Frinps. C. S.: Foster. L. B. Am. J. Clin. Pathol. 1970.53.944. 5. Chri;tensen,'H. Act; Pharmacol. Tozicol. 1977, 40, 289. ' 6. Nash, J. F.; Bennett, I. F.; Bopp, R. J.; Brunson, M. K.; Sullivan, H. R. J. Pharm. Sci. 1975,64,429. 7. Walle, T.; Ehrsson, H. Acta Pharm. Succica 1971, 8 , 27. 8. Cleemann, M. J. Chromatogr. 1977, 132,287. 9. Angelo, H. R.; Christensen, J. M. J. Chromatogr. 1977, 140, 280. 10. Manno, J.; Jain, N.; Crim, D.; Forney, R. J . Forensic Sci. 1970, 15, 403. 11. Wolen, R. L.; Gruber, C. M., Jr. Anal. Chem. 1 9 6 8 , 4 0 , 1243. 12. Verebely, K.; Inturrisi, C. E. J. Chromatogr. 1973, 75, 195. 13. Millard, B. J.; Sheinin, E. B.; Benson, W. R. J. Pharm. Sci. 1980, 69. 1177. 14. Sparacino, C. M.; Pellizzari, E. D.; Cook, C. E.; Wall, M. W. J . Chromatogr. 1973, 77, 413. 15. Gilpin, R. K.; Korpi, J. A.; Janicki, C. A. J . Chromatogr. 1975, -707 - . , -115 - -. 16. Borkan, S.; Wainer, I. W. J . Chromatogr. 1982, 240, 547. 17. Kunka, R. L.; Venkataramanan, R.; Stern, R. M.; Ladik, C. F. Clin. Pharmacol. Ther. 1 9 8 4 , 3 5 , 675.
Acknowledgments Supported in part by a grant from the Magee Womens Hospital Research Fund, Pittsburgh, PA. The authors thank Ms. Judy Cherevka for manuscript preparation and Ms. Rhonda Harrison for technical assistance.