Page 43 - Modern Derivatization Methods for Separation Sciences

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permit highly sensitive and selective measurement of analytes in biological samples without tedious
clean-up or derivatization steps.

On an economical basis, although LC-MS/MS instruments are quite expensive, the total analytical cost
is almost similar or less compared with conventional HPLC methods owing its high sample throughput
with less man-power. In addition, guidelines for new drug applications world-wide, require high
reliability of analytical methods together with Good Laboratory Practice standards. From this point of
view, HPLC analytical methods with derivatization and/or clean-up steps have disadvantages not only
in sensitivity and selectivity, but also sample through put and reliability. On reliability, LC-MS and
LCMS/MS can utilize the ultimate internal standard, i.e. stable isotope labeled analogue.

In LC-MS/MS operations, APCI covers lower to middle polar compounds and ESI covers middle to
high polar ones. However, highly watersoluble compounds sometimes prove difficult in sensitive
measurement even if ESI is employed. These compounds are extracted with difficulty and concentrated
from the biological matrix. Demands for novel derivatization reagents/methods for these types of
compounds are increasing, especially for those which are reactive in a water matrix.

For enantiospecific determination of drugs, HPLC methods using chiral derivatization reagents still has
advantages over other techniques. However, it is difficult for selective determination of a drug that has
two or more chiral centers in the molecule. We have encountered a compound that has three chiral
centers, i.e. eight optical isomers. The compound is an active metabolite of CS-670, a non-steroidal
anti-inflammatory agent, that is a derivative of 2-phenylpropionate, the same group as ibuprofen. In this
case, one chiral center has separated using immunoaffinity purification followed by enantioselective
derivatization to trace the active metabolite level in plasma [38].

There are many racemate drugs in market and some of those are half active and half inactive.
Regulations require clarification of the precise disposition of an individual enantiomer in a body
including its toxicity and pharmacokinetic

properties. See the recent reviews in this field [39,40]. In addition, derivatized enantiomers should be
detected using high sensitivity and specificity methods, such as LC-MS/MS.

References

[1] Tsujita, Y., Kuroda, M., Shimada, Y., Tanzawa, K., Arai, M., Kaneko, I., Tanaka, M., Masuda,
H.,Tarumi, C., Watanabe, Y. and Fujii, S. (1986) Biochim. Biophys. Acta, 877, 50.

[2] Arai, M., Serizawa, N., Terahara, A., Tsujita, Y., Tanaka, M., Masuda, H. and Ishikawa, S. (1988)
Ann. Rep. Sankyo Res. Labs, 40, 1.

[3] Dumousseaux, C., Muramatsu, S., Takasaki, W. and Takahagi, H. (1994) J. Pharm. Sci. 83, 1630.

[4] Glencross, R.G., Abeywardene, S.A., Corney, S.J. and Morris, H.S. (1981) J. Chromatogr., 223,
193.

[5] Takahagi, H., Inoue, K. and Horiguchi, M. (1986) J. Chromatogr., 352, 369.

[6] Caldwell, J., Hutt, A.J. and Fournel-Gigleux, S. (1988) Biochem. Pharmacol. 37, 105.

[7] Foster, R.T. and Jamali, F. (1987) J. Chromatogr. 416,388.

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