We report on the utility of urine total buprenorphine, total norbuprenorphine, and creatinine concentrations in patients treated with Suboxone (a formulation containing buprenorphine and naloxone), used increasingly for the maintenance or detoxification of patients dependent on opiates such as heroin or oxycodone. Patients received 8-24 mg/day buprenorphine. Two-hundred sixteen urine samples from 70 patients were analyzed for both total buprenorphine and total norbuprenorphine by liquid chromatography-mass spectrometry (LC-MS-MS). Buprenorphine concentrations in all 176 samples judged to be unadulterated averaged 164 ng/mL, with a standard deviation (SD) of 198 ng/mL. Nine samples (4.2%) had metabolite-parent drug ratios < 0.02, and 33 (15.3%) had no detectable buprenorphine. The metabolite/parent drug ratio in 166 samples had a range of 0.07-23.0 (mean = 4.52; SD = 3.97). Fifteen of 96 available urine samples (16.7%) had creatinine less than 20 mg/dL. We also found sample adulteration in 7 (7.3%) available samples. Using a 5 ng/mL urine buprenorphine cutoff, the sensitivity and specificity of the Microgenics homogeneous enzyme immunoassay versus LC-MS-MS were 100% and 87.5%, respectively. The 5 ng/mL cutoff Microgenics CEDIA buprenorphine assay results agreed analytically with LC-MS-MS in 97.9% of samples.
sion spectra. Thus, we used dNTPs labeled with Cy5, Cy3, and Alexa 594, which have distinct spectra. Accordingly, our CDH results showed improved microarray imaging because there was less nonspecific hybridization ( Fig. 1). We also noted that the wild-type (codon 12 and 13) signals were slightly reduced. This can be explained by the fact that the digested wild-type DNA from each sample had to compete with each other for hybridization. In contrast, the mutant DNA, which rarely overlapped in the three mixed samples, did not compete and thus produced a strong signal. As a result, when the sample had a mutation, the signal ratios between mutant and wild-type DNA increased from 0.91 to 1.66 (Fig. 1, A 1, 8 ). Samples can be scored as containing a mutation when the ratio is above the threshold; therefore, the larger ratio can make mutational analysis more correct. (c) Mixing of samples reduces experimental cost and time. In addition, each K-ras microarray contained three separate oligonucleotide sets. This allows researchers to investigate nine samples per microarray, facilitating large-scale analysis. We hybridized three different DNA samples into which the fluorescently labeled dNTPs had been incorporated in one experiment to detect K-ras mutations by the K-ras oligonucleotide microarray. The CDH technology increased the discrimination of mutation signal from nonspecific signals (Fig. 1).In summary, we developed a K-ras oligonucleotide microarray and applied our new CDH method to identify 50 K-ras mutations in samples from 204 Korean colorectal cancer patients. The results of the K-ras microarray analysis agreed perfectly with conventional sequencing data. The new method increases efficiency through pooling of multiple samples, providing a sensitive, rapid, highthroughput system that may be suitable for large-scale studies that require simple, quick, and effective screening of large numbers of samples.
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