Improved high-performance liquid chromatographic method for the determination of ethylmorphine and its metabolites in microsomal incubations and cell culture media.

Ethylmorphine N-demethylation is used as a marker pathway in studies of rat cytochrome P450 3A and 2C11 biotransformations. At present, microsomal activities are generally measured by a colorimetric determination of the formed formaldehyde. In the present study, a high-performance liquid chromatographic method of separating and quantifying both the N-demethylated (norethylmorphine) and the O-de-ethylated (morphine) metabolites is described. Either samples are extracted with ethyl acetate or proteins are precipitated with zinc sulphate-barium hydroxide. Separation is achieved on a CN reversed-phase column, using a mobile phase of phosphate buffer (pH 4.5)-acetonitrile (90:10, v/v). At a flow-rate of 1.5 ml/min, the analysis time is 30 min. The limit of detection (ultraviolet, 210 nm) for ethylmorphine and its metabolites is 0.5 micrograms/ml.     

Measuring the threshold for speech reception by adaptive variation of the signal bandwidth. II. Hearing-impaired listeners

In a previous study [Noordhoek et al., J. Acoust. Soc. Am. 105, 2895-2902 (1999)], an adaptive test was developed to determine the speech-reception bandwidth threshold (SRBT), i.e., the width of a speech band around 1 kHz required for a 50% intelligibility score. In this test, the band-filtered speech is presented in complementary bandstop-filtered noise. In the present study, the performance of 34 hearing-impaired listeners was measured on this SRBT test and on more common SRT (speech-reception threshold) tests, namely the SRT in quiet, the standard SRT in noise (standard speech spectrum), and the spectrally adapted SRT in noise (fitted to the individual's dynamic range). The aim was to investigate to what extent the performance on these tests could be explained simply from audibility, as estimated with the SII (speech intelligibility index) model, or require the assumption of suprathreshold deficits. For most listeners, an elevated SRT in quiet or an elevated standard SRT in noise could be explained on the basis of audibility. For the spectrally adapted SRT in noise, and especially for the SRBT, the data of most listeners could not be explained from audibility, suggesting that the effects of suprathreshold deficits may be present. Possibly, such a deficit is an increased downward spread of masking.     

Measuring the threshold for speech reception by adaptive variation of the signal bandwidth. I. Normal-hearing listeners.

An adaptive test has been developed to determine the minimum bandwidth of speech that a listener needs to reach 50% intelligibility. Measuring this speech-reception bandwidth threshold (SRBT), in addition to the more common speech-reception threshold (SRT) in noise, may be useful in investigating the factors underlying impaired suprathreshold speech perception. Speech was bandpass filtered (center frequency: 1 kHz) and complementary bandstop filtered noise was added. To obtain reference values, the SRBT was measured in 12 normal-hearing listeners at four sound-pressure levels, in combination with three overall spectral tilts. Plotting SRBT as a function of sound-pressure level resulted in U-shaped curves. The most narrow SRBT (1.4 octave) was obtained at an A-weighted sound-pressure level of 55 dB. The required bandwidth increases with increasing level, probably due to upward spread of masking. At a lower level (40 dBA) listeners also need a broader band, because parts of the speech signal will be below threshold. The SII (Speech Intelligibility Index) model reasonably predicts the data, although it seems to underestimate upward spread of masking.     

Immunochemical detection of metabolites of parent and nitro polycyclic aromatic hydrocarbons in urine samples from persons occupationally exposed to diesel exhaust

An immunochemical assay was developed for the detection of metabolites excreted in urine as a result of occupational exposure to PAHs and nitro-PAHs. These metabolites were analyzed in a competitive ELISA, using an existing antibody (4D5) that has been developed against 6-aminobenzo[a]pyrene coupled to bovine serum albumine (B[a]P-BSA). A solid phase extraction (SPE) work-up procedure was optimized by dilution of pooled urine samples from rats exposed to 1-NP, in human urine. The application was validated by comparison of test results with urinary excreted 1-hydroxypyrene (1-OH-P) levels in a study among 28 railroad workers. Excretion of urinary metabolites was determined over two consecutive workdays. The 24 h average excretion of metabolites as determined in the immunoassay was not related to levels of particulate matter in the breathing zones of workers, not to 1-OH-P excretion levels of the day of urine collection. However, it was significantly associated with the personal dust exposure of the day before (P<0.0001), suggesting slow excretion of urinary metabolites. Smoking habits caused minor interference (P<0.1).     

Determination of trimethoprim and its oxidative metabolites in cell culture media and microsomal incubation mixtures by high-performance liquid chromatography.

A high-performance liquid chromatographic method is presented for the determination of trimethoprim (TMP), 3'-hydroxy-TMP, 4'-hydroxy-TMP, alpha-hydroxy-TMP and two TMP N-oxides. The last two metabolites appear to decompose on liquid extraction. TMP and its oxidative metabolites are separated using a C18 radial-compression column and quantified by UV detection at 230 nm. Calibration curves are linear from 0.5 to at least 50 microM. The limit of detection is 0.05-0.15 micrograms/ml. In in vitro rat liver metabolism studies. 3'- and 4'-hydroxylation of TMP appear to be important metabolic pathways whereas TMP N-oxides are minor metabolites.