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.

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-

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

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