4.2 © Springer-Verlag Berlin Heidelberg 2005 II.4.2 Propionic acid derivative analgesic-antipyretics by Tatsuo Shinozuka and Rika Nakajima Introduction Propionic acid derivative analgesic-antipyretics (> Table 2.1) are non-steroidal and anti- in ammatory drugs. As one of mechanisms of their pharmacological actions, inhibition of prostaglandin biosynthesis can be mentioned. Although fatal cases due to propionic acid derivative analgesic-antipyretics are not many in the world, the incidence of poisoning (including survived cases) by this drug group is relatively high among therapeutic drugs in Japan [1]. Analyses of propionic acid derivative analgesic-antipyretics are being made by TLC [2, 3], HPLC [2, 4–8], GC [9], GC/MS [10] and LC/MS [11]. In this chapter, the methods of analysis of this drug group by TLC, HPLC and GC are presented. TLC analysis Reagents and their preparation • Ibuprofen, ketoprofen, naproxen, fenoprofen calcium,  urbiprofen, loxoprofen and oxa- prozin can be purchased from Sigma (St. Louis, MO, USA). For other drugs, pure powder of each drug can be obtained by direct request to each manufacturer as follows: alminopro- fen from Maruho Pharmaceutical Co., Ltd., Tokyo, Japan; zaltoprofen from Japan Chemi- phar Pharmaceutical Co., Ltd., Tokyo, Japan; thiaprofenic acid from Aventis Pharma, Stras- bourg, France; pranoprofen from Mitsubishi Welpharma, Osaka, Japan. • High-performance (HP) TLC plate a : silica gel 60 F 254 HPTLC (Merck, Darmstadt, Germany). • Dichloroindophenol reagent: 0.05 g of 2,6-dichloroindophenol sodium (Sigma) is dis- solved in 50 mL ethanol. • Bromocresol green reagent: 0.04 g of bromocresol green sultone form (Sigma) is dissolved in 100 mL of 96 % ethanol. • Dragendor reagent: 0.85 g of bismuth subnitrate is dissolved in a mixture of 40 mL dis- tilled water and 10 mL acetic acid to prepare “A” solution. A 8-g aliquot of potassium iodide is dissolved in 20 mL distilled water to prepare “B” solution.  en, a mixture of A/B/acetic acid/distilled water (1:1:4:20, v/v) is prepared. • Ninhydrin reagent: 0.2 g of ninhydrin is dissolved in a mixture of 5 mL acetic acid and 95 mL n-butanol. • Liebermann’s reagent: 2 g of sodium nitrite is dissolved in 20 mL of concentrated sulfuric acid. 326 Propionic acid derivative analgesic-antipyretics ⊡ Table 2.1 Structures of propionic acid derivative analgesic-antipyretics alminoprofen ibuprofen oxaprozin ketoprofen zaltoprofen tiaprofenic acid naproxen fenoprofen calcium pranoprofen flurbiprofen loxoprofen sodium 327 Developing solvents Benzene/acetone (3:2, v/v). n-Butyl ether/n-hexane/acetic acid (20:4:1, v/v). Chloroform/methanol/acetic acid (45:5:1, v/v). Procedure i. A 0.1-mL volume of urine or serum (plasma) is mixed with 0.9 mL of 0.2 M disodium hydrogenphosphate/0.1 M citric acid bu er solution (pH 3.0), and extracted with 1 mL chloroform three times. ii.  e combined chloroform extract is mixed well with 0.5 g of anhydrous sodium sulfate to dehydrate it and passed through  lter paper; the  ltrate is evaporated to dryness under reduced pressure. iii.  e residue is dissolved in a small amount of methanol to serve as a test solution. iv. At a location 1-cm up from the bottom of a TLC plate, the above organic extract is spotted with a size of 1–2 mm diameter. A er drying the spot, the plate is placed in a development tank  lled with vapor of a developing solvent and the spot is developed with the developing solvent. v. A er development, the plate is dried with a blower, and the  uorescence is observed under ultraviolet light at 365 nm; light absorbing spots are also observed under the light at 254 nm. A er the above observations, the plate is sprayed with each reagent.  e tentative identi - cation is made by spotting the authentic standard together with the test extract. A er spraying the dichloroindophenol reagent, the TLC plate should be heated at 100 °C to de- tect spots. Assessment of the method > Table 2.2 shows R f values of eleven propionic acid derivative analgesic-antipyretics for HPTLC (normal phase) with three solvent systems. > Table 2.3 shows the detection limits and colors of the spots observed under ultraviolet light and a er spraying  ve kinds of reagents.  e screening by TLC is simple and rapid for unchanged drugs. Reversed phase TLC for drug analysis was also reported [2, 3].  e optimization of a solvent system for drug analysis by TLC can give a useful hint for preparing a mobile phase of HPLC or LC/MS. HPLC analysis Reagents •  e sources of drugs are the same as speci ed in the section of TLC analysis. N-Chloromethyl- phthalimide (N-CMPI) can be purchased from Sigma. • Oasis ® MAX cartridges b were obtained from Waters (Milford, MA, USA). HPLC analysis 328 Propionic acid derivative analgesic-antipyretics ⊡ Table 2.2 R f values of propionic acid derivative analgesic-antipyretics obtained by high-performance (HP) TLC Compound Developing solvents 123 alminoprofen 0.58 0.30 0.64 ibuprofen 0.67 0.53 0.67 oxaprozin 0.66 0.23 0.61 ketoprofen 0.42 0.32 0.64 zaltoprofen 0.66 0.34 0.63 tiaprofenic acid 0.65 0.29 0.61 naproxen 0.57 0.40 0.66 fenoprofen calcium 0.57 0.47 0.67 pranoprofen 0.36 0.03 0.59 flurbiprofen 0.51 0.46 0.65 loxoprofen sodium 0.52 0.23 0.64 1. benzene/acetone (3:2, v/v). 2. n-butyl ether/n-hexane/acetic acid (20:4:1, v/v). 3. chloroform/methanol/acetic acid (45:5:1, v/v). ⊡ Table 2.3 Colors and detection limits of spots of propionic acid derivative analgesic-antipyretics observed by HPTLC Componnd Detection limits (µg) (color) 3 4 5 6 7 Ultraviolet light alminoprofen 1 (light orange) 1 (yellowish green) – – 1 (dark brown) 0.1 ibuprofen 5 (pink) – – – – 2 oxaprozin 1 (light orange) 1 (yellow) 1 (orange) – 1 (brown) 0.1 ketoprofen 1 (pink) – – – 1 (dark gray) 0.1 zaltoprofen 1 (light orange) 1 (yellow) 1 (orange) – 1 (dark brown) 0.1 (fluorescent) tiaprofenic acid 1 (light orange) 1 (yellow) 1 (orange) – 1 (brown) 0.1 naproxen 1 (pink) 1 (yellow) 1 (orange) – 0.5 (yellowish brown) 0.1 (fluorescent) fenoprofen calcium 1 (pink) 2 (yellowish green) – – 1 (brown) 1 pranoprofen 1 (pink) 1 (yellowish green) 1 (orange) – – 0.1 (fluorescent) flurbiprofen 1 (pink) – – – 1 (yellowish brown) 0.1 loxoprofen sodium 5 (pink) – – – 1 (dark gray) 5 3. dichloroindophenol reagent. 4. bromocresol green reagent. 5. Dragendorff reagent. 6. ninhydrin reagent. 7. Liebermann’s reagent. 329 Analytical conditions Instrument: a Hitachi HPLC (L-6200) instrument equipped with a UV detector (L-2400) (Hitachi Ltd., Tokyo, Japan); column: LiChrospher RP-18 (e) (250 × 4.0 mm i.d., Merck, Darm- stadt, Germany); mobile phase: methanol/puri ed water (70:30, v/v); its  ow rate: 0.5 mL/min; detection wavelength: 293 nm. Procedures • Preparation-1: the extracts obtained by the procedure i–iii of the TLC analysis section of this chapter are condensed or diluted before instrumental analysis. • Preparation-2: solid-phase extraction with an Oasis ® MAX cartridge is made as follows: i. A 0.5-mL volume of urine or serum (plasma) is mixed well with 0.1 mL phosphoric acid and 0.5 mL of puri ed water and poured into an Oasis ® MAX cartridge, which had been activated by passing 3 mL methanol and 3 mL puri ed water. ii. All manipulations are made using a Vac-Elut ® system (Varian, Harbor City, CA, USA) with aspiration pressure at 15–20 mmHg. iii.  e cartridge is washed with 2 mL of 50 mM sodium acetate solution containing 5 % meth- anol, and drugs are eluted with 3 mL of 100 mM phosphoric acid solution/acetonitrile (20:80, v/v). iv.  e eluate is evaporated to dryness, and the residue is dissolved in 100 µL methanol con- taining n-caprylic acid as IS. v. A 50-µL volume of the above solution, 50 µL of 100 mM N-CMPI acetonitrile solution, 50 µL of 100 mM triethylamine acetonitrile solution are placed in a screw cap test tube (10 × 1.5 cm) and mixed well. vi.  e mixture tube is heated at 80 °C for 30 min in a water bath or on an aluminum block heater. A er cooling to room temperature, a 1–3 µL aliquot of it is injected into HPLC. vii. Various concentrations c of a target drug are spiked into blank specimens, and processed according to the above procedure to construct a calibration curve; the IS is also added at the step iv.  e peak area ratio of a test compound to IS is applied to the calibration curve to obtain its concentration. Assessment of the method > Figure 2.1 shows an HPLC chromatogram for methylphthalimide (MPI) derivatives of eight propionic acid derivative analgesic-antipyretics d .  e retention times and detection limits of the drugs obtained by this method are shown in > Table 2.4. HPLC analysis 330 Propionic acid derivative analgesic-antipyretics HPLC chromatogram for methylphthalimide (MPI) derivatives of propionic acid derivative analgesic-antipyretics extracted from human blood [4]. 1: pranoprofen; 2: loxoprofen; 3: ketoprofen; 4: naproxen; 5: fenoprofen; 6: flurbiprofen; 7: alminoprofen; 8: ibuprofen; IS: n-caprylic acid; the concentration of each drug spiked into blood was 10 µg/mL. ⊡ Figure 2.1 ⊡ Table 2.4 Retention times and detection limits of MPI derivatives of propionic acid derivative analgesic- antipyretics obtained by HPLC-UV Compound Retention time (min) Detection limit (injected amount) (ng) alminoprofen 37.3 40.0 ibuprofen 42.0 50.0 ketoprofen 14.0 16.7 naproxen 18.7 9.6 fenoprofen calcium 25.6 22.2 pranoprofen 10.0 8.7 flurbiprofen 29.4 44.4 loxoprofen sodium 12.0 23.5 331 GC analysis Reagents and their preparation •  e sources of drugs are the same as speci ed in the TLC analysis section. • Methylation reagent: phenyltrimethylammonium hydroxide ( PTAH) methanolic solution e (20–25 %, about 1.3 M, Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) is diluted 26-fold with methanol to prepare 50 mM PTAH just before use. • 9-Anthracenecarboxylic acid (Sigma) is dissolved in puri ed water to prepare 500 µg/mL solution to be used as IS solution. • Bond Elut ® SI column f (Analytichem International, Harbor City, CA, USA). GC conditions Instrument: a Shimadzu GC-14A gas chromatograph with an FID (Shimadzu Corp., Kyoto, Japan); column: CBP-1 (25 m × 0.33 mm i. d.,  lm thickness 0.5 µm, Shimadzu Corp.); injec- tion temperature: 280 °C g ; column (oven) temperature: 100 °C → 3 °C/min → 280 °C; detector temperature: 320 °C; carrier gas ( ow rate): He (5 mL/min); make-up gas ( ow rate): He (30 mL/min); injection: splitless mode. Procedure i. A 0.1-mL volume of urine or serum (plasma) is mixed with 0.9 mL of 0.2 M disodium hydrogenphosphate/0.1 M citric acid bu er solution (pH 3.0), and extracted with 2 mL chloroform 3 times. ii.  e combined chloroform extract is condensed to about 1 mL, and poured into a Bond Elut ® SI column. iii.  e column is washed with 6 mL of diethyl ether/hexane (1:1, v/v) and the drugs are eluted with 6 mL of chloroform/methanol/acetic acid (45:5:1, v/v). iv. A 5-µL aliquot of IS (9-anthracenecarboxylic acid) solution (containing 2.5 µg of the compound) is added to the eluate, and evaporated to dryness under reduced pres- sure. v.  e residue is dissolved in 25 µL of 50 mM PTAH methanolic solution and a 1–2 µL ali- quot of it is injected into GC. vi.  e quantitation is made with a calibration curve, which had been constructed using vari- ous concentrations h of a target drug and a  xed amount of IS. Assessment of the method > Figure 2.2 shows a gas chromatogram for ten propionic acid derivative analgesic-antipyret- ics using on-column methylation i .  e retention times and detection limits obtained by this method are shown in > Table 2.5. Maurer et al. [10] have reported GC/MS analysis of 40 GC analysis 332 Propionic acid derivative analgesic-antipyretics ⊡ Figure 2.2 Gas chromatogram for methyl derivatives of propionic acid derivative analgesic-antipyretics extracted from human blood plasma [9]. 1: ibuprofen; 2: alminoprofen; 3: tiaprofenic acid; 4: fenoprofen; 5: flurbiprofen; 6: naproxen; 7: loxoprofen; 8: ketoprofen; 9: pranoprofen; 10: zaltoprofen; IS: 9-anthracenecarboxylic acid; the concentration of each drug spiked into plasma was 2–20 µg/mL. ⊡ Table 2.5 Retention times and detection limits of methyl derivatives of propionic acid derivative analgesic- antipyretics obtained by GC-FID Compound Retention time (min) Detection limit (µg/mL) alminoprofen 25.8 1.0 ibuprofen 15.6 0.2 ketoprofen 35.3 0.2 zaltoprofen 49.4 1.0 tiaprofenic acid 27.6 1.0 naproxen 31.7 0.5 fenoprofen calcium 28.3 0.2 pranoprofen 41.8 2.0 flurbiprofen 28.9 0.5 loxoprofen sodium 32.4 2.0 333 analgesic-antipyretics (including ibuprofen, ketoprofen, tiaprofenic acid, fenoprofen, naproxen and  urbiprofen) with methyl derivatization. Poisoning cases, blood concentrations and fatal doses Case 1 [12]: a 49-year-old female died during admission to a hospital for treatments of bone fracture of the right leg in icted by a tra c accident. She had a past history of bronchial asthma. It was estimated that she had died of asthmatic attack due to ketoprofen shock.  e concentration of ketoprofen in blood sampled at autopsy was 160 ng/mL, and that in the liver was 10 ng/g. Case 2 [13]: a 37-year-old male died during being mentally deranged. It was disclosed that he had ingested 9 tablets (675 mg) of ibuprofen.  e action of ibuprofen on the central nervous system was estimated to be related with the cause of his death.  e oral LD 50 values obtained from mice for eleven propionic acid derivative analgesic- antipyretics (alminoprofen, ibuprofen, oxaprofen, ketoprofen, zaltoprofen, tiaprofenic acid, naproxen, fenoprofen calcium, pranoprofen,  urbiprofen and loxoprofen sodium) were re- ported to be 400–1,500 mg/kg [14].  e blood concentrations of drugs of this group described in literature [15] are as follows. Feneprofen: therapeutic range, 23–31 µg/mL, a patient recovered even a er ingestion of as much as 60 g fenoprofen;  urbiprofen: therapeutic range, 9.1–16.6 µg/mL; ibuprofen: thera- peutic range, 18–24 µg/mL, a patient ingested 12 g ibuprofen to attempt suicide, fell into a co- matose state with its blood level at 840 µg/mL, but recovered within 24 h; ketoprofen: thera- peutic range, 4.7–14.3 µg/mL; naproxen: therapeutic range, 26–69 µg/mL, a patient ingested 25 g naproxen, showed its blood level at 414 µg/mL a er 15 h, but recovered; tiaprofenic acid: therapeutic range, 18.6–73.3 µg/mL. Notes a) In this method, high-performance (HP) TLC plates were used.  ey give high resolution and 10–50 times lower detection limits, and are suitable for small amounts of specimens. However, usual TLC plates can be also used for the present analysis. b)  e Oasis ® MAX cartridges are commercially available for solid-phase extraction of neutral and acidic compounds in biomedical specimens such as serum and urine.  e cartridge is suitable for pretreatments before analysis by HPLC and LC/MS [11]. c) Solutions of each drug at 1–10 µg/mL are prepared. d)  ere are many reports dealing with HPLC analysis of analgesic-antipyretics including the propionic acid drugs without any derivatization [2, 5–8]. However, the derivatization with N-CMPI results in increase of sensitivity several ten times for HPLC analysis with a UV detector. Recently, the authors have reported a method for LC/MS analysis of the pro pionic acid drugs without any derivatization [11]. e) Every propionic acid derivative analgesic-antipyretic listed in Table 2.1 has a carboxylic acid group and is very suitable for the on-column methylation. As on-column methylating reagents, phenyltrimethylammonium hydroxide (trimethylanilinium hydroxide) (PTAH) and tetramethylammonium hydroxide ( TMAH) are being sold; the former reagent gives better results for the present propionic acid derivative drugs. Poisoning cases, blood concentrations and fatal doses 334 Propionic acid derivative analgesic-antipyretics f)  e column should be activated by passing 10 mL methanol before use. g) When the injection temperature is not higher than 200 °C, the e ciency of on-column methylation becomes much lower. h) Solutions of each drug at 1–10 µg/mL are prepared. i)  e propionic acid derivative drugs cannot be analyzed by GC without derivatization. References 1) Japan Poison Information Center (2001) 2000 Annual report by JPIC. Jpn J Toxicol 14:145–164 (in Japanese) 2) Shinozuka T, Terada M (1995) Standard methods of chemical analysis in poisoning VII, analgesics and anti- pyretics, the 115th Annual Meeting of the Pharmaceutical Society of Japan, the data of the Public Health Council. Pharmaceutical Society of Japan, Tokyo, pp 81–86 (in Japanese) 3) Shinozuka T, Terada M, Ogamo A et al. (1996) Data on high-performance thin-layer chromatography of analgesic and antipyretic drugs. Jpn J Forensic Toxicol 14:246–252 4) Shinozuka T, Takei S, Kuroda N et al. (1996) Simultaneous determination of propionic acid analgesic and anti- pyretic drugs in human blood and urine by high-performance liquid chromatography. Jpn J Forensic Toxicol 14:43–51 5) Shinozuka T, Terada M, Ogamo A et al. (1997) Simultaneous determination of analgesic-antipyretic drugs by high-performance liquid chromatography in the human serum. Proceedings of the 14th Meeting of the Inter- national Association of Forensic Science, Vol. 2. Shunderson Communications, Ottawa, pp 227–230 6) Battista HJ, Wehinger G, Henn R (1985) Separation and identification of non-steroidal antirheumatic drugs containing a free carboxyl function using high-performance liquid chromatography. J Chromatogr 345:77–89 7) Moore CM, Tebbett IR (1987) Rapid extraction of anti-inflammatory drugs in whole blood for HPLC analysis. Forensic Sci Int 34:155–158 8) Owen SG, Roberts MS, Friesen TW (1987) Rapid high-performance liquid chromatographic assay for the simul- taneous analysis of non-steroidal anti-inflammatory drugs in plasma. J Chromatogr 416:293–302 9) Shinozuka T, Nakajima R, Takei S et al. (1999) Simultaneous analysis of propionic acid analgesic and antipyretic drugs in human plasma by gas chromatography. Jpn J Forensic Toxicol 17:51–60 10) Maurer HH, Tauvel AX, Kraemer T (2001) Screening procedure for detection of non-steroidal anti-inflammatory drugs and their methabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography- mass spectrometry after extractive methylation. J Anal Toxicol 25:237– 244 11) Shinozuka T, Nakajima R, Ohue O et al. (2001) Analysis of analgesic and antipyretic drugs by LC/MS. Jpn J Forensic Toxicol 19:172–173 (in Japanese with an English abstract) 12) Moriya F, Hashimoto Y (1996) A case of fatal asthmatic attack probably induced by ketoprofen disclosed after screening of non-steroidal anti-inflammatory drugs. Jpn J Forensic Toxicol 14:240–245 13) Yanai A, Seo Y, Kakizaki E et al. (1998) An autopsy case in which ibuprofen poisoning was estimated. Res Pract Forensic Med 41:313–317 (in Japanese with an English abstract) 14) Yamazaki F, Mori H (eds) (2000) Guide to Acute Poisonings by Medical Drugs. Van Medical, Tokyo, pp 82–83 (in Japanese) 15) Moffat AC, Jackson JV, Moss MS et al. (eds) (1986) Clarke,s Isolation and Identification of Drugs, 2nd edn. The Pharmacautical Press, London, pp 615–616; 631–632; 677–678; 697–698; 799–800; 1024 . acid derivative analgesic-antipyretics (> Table 2.1) are non-steroidal and anti- in ammatory drugs. As one of mechanisms of their pharmacological actions,. Society of Japan, Tokyo, pp 81–86 (in Japanese) 3) Shinozuka T, Terada M, Ogamo A et al. (1996) Data on high-performance thin-layer chromatography of analgesic