Different kinetics of bone markers in normal and delayed fracture healing of long bones.

that both gabapentin and the internal standard are recovered from plasma in a 90 –98% yield after extraction with 1-mL C 18 extraction columns. The recovery of gaba-pentin is drastically reduced when the sample passes through the C 18 sorbent at a relatively fast rate. However, endogenous amino compounds present in plasma are more polar than gabapentin and are not retained by C 18 sorbent even when the sample is passed at a slow rate. Different authors have used different compositions of OPA derivatizing agent, a mixture of a methanolic solution of OPA, a thiol, and borate buffer, but the fluorescent derivatives are produced in similar yields. The presence of water in the reagent makes the OPA derivative unstable , and the stability can be improved by decreasing the water content (16). In our described procedure, the deriv-atizing reagent does not contain borate buffer. The meth-anolic eluate obtained from the C 18 extraction column contains traces of borate buffer, which is adequate to initiate the reaction. The resulting OPA derivatives are stable for at least 4 h at room temperature and for at least 24 h when stored at 4 – 8 °C. Gabapentin OPA derivatives have been detected at excitation wavelengths of 230 –250 or 330 –350 nm with a common emission wavelength of 430 – 450 nm. Excitation at 230 –250 nm provides ϳ2.5 times the sensitivity of that observed at 330 –350 nm with the same attenuation, slit width, and emission wavelength. However, the baseline is unstable at excitation wavelengths of 230 –250 nm, and the detector must be set at a higher attenuation to decrease the noise. The procedure presented here is extremely sensitive even at an excitation wavelength of 350 nm. ␣-Aminocyclohexanepropionic acid (cat. no. 43,805-7; Aldrich) behaved similar to gabapentin and its analog for solid-phase extraction. It produced a fluorescent OPA derivative that eluted close to the gabapentin analog commonly used as the internal standard. However, the fluorescence response of this compound was much weaker than that of gabapentin, and it had to be used at a concentration of 50 mg/L. This did not affect the linearity of the procedure. We thank Dr. A. Fraser of Victoria General Hospital, Halifax, and Pfizer Canada for supplying us with samples of gabapentin and 1-(aminomethyl)cycloheptane acetic acid. Gabapentin quantification in human plasma by high performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bio-equivalence study. …