γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter having anti-anxiety, stress-reducing and sleep-enhancing effects. The blood GABA concentration has the potential to become a diagnostic index for depression and schizophrenia. In this study, we developed a method for the quantification of GABA in human serum by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using a pair of reagents, 4-diethylaminobenzoic acid N-succinimidyl ester (DEABAS) and its deuterated isotopologue (dDEABAS). The deproteinized serum samples were derivatized with DEABAS, to which the dDEABAS-derivatized standard GABA of known amount was then added. The DEABAS-derivatization enhanced not only the ESI-MS/MS sensitivity but also the LC retention, which enabled the separation of GABA from the interfering isomers. Satisfactory assay precision and accuracy were also acquired by adding dDEABAS-derivatized GABA, which served as the internal standard and worked well for correcting the matrix effect and instrument drift. The developed method had satisfactory applicability to the real sample analysis because it was successfully used to quantify the serum GABA of the healthy subjects.
A fully-automated two-dimensional (2D) HPLC system for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers was developed. The system was validated and applied to the determination of the target hydroxy amino acid enantiomers in vinegar samples including the Japanese traditional amber rice vinegar. The Japanese traditional amber rice vinegar is one of the typical natural fermented products containing high levels of D-amino acids, and the clarification of the amounts of hydroxy amino acid enantiomers is expected. For the on-line 2D-HPLC system, a narrowbore ODS column (Singularity RP18, 1.0 x 250 mm) was used in the first dimension, and an originally-designed Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 x 250 mm) was utilized in the second dimension. The target hydroxy amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence (ex. 470 nm, em. 530 nm). The concentrations and the %D values were compared among a grain vinegar, a widely-distributed black vinegar and Japanese traditional amber rice vinegars (fermented/aged 1 or 5 years by microorganisms in an earthenware jar). The amounts of hydroxy amino acid enantiomers were low (0.21-0.38 μmol/mL, only L-forms) in the grain vinegar, and relatively high amounts of the L-forms were observed (1.05-1.61 μmol/mL) in the widely-distributed black vinegar. On the other hand, clear peaks of the D-forms (0.16-0.86 μmol/mL for D-Ser, 0.02-0.11 μmol/mL for D-aThr) as well as high amounts of the L-forms (2.27-2.51 μmol/mL for L-Ser, 1.85-2.10 μmol/mL for L-Thr) were observed in the Japanese traditional amber rice vinegars. The amounts of D-Ser and D-aThr clearly increased with increasing the fermentation/aging period. This is the first report showing the profiles of the hydroxy amino acid enantiomers simultaneously, and further investigations using various fermented food/beverage samples are expected.
This fundamental study was conducted to increase the multiplicity of frequency division in multiplex high-performance liquid chromatography-mass spectrometry (HPLC-MS). Virtual chromatograms were used to investigate the high efficiency separation, i.e., the peak with a narrow band in time-domain chromatograms. They revealed that the peak induces the broad signals in the frequency-domain power spectrum obtained from the Fourier transform (FT) of the chromatogram. For a high performance separation of N = 50 000 and 200 000, a minimum modulation frequency of 22.5 and 45 Hz, respectively, are required for 4HPLC-1MS. The high-frequency modulation was successfully achieved by increasing discharge rate with modifying the electric circuit in the modulation system. In addition, a higher sampling rate in MS is required for a higher modulation frequency to achieve successful FT analytical results. In other words, the sampling frequency must be increased by a factor of five for a given modulation frequency. Using a laboratory-made four channel interface, the simultaneous sample introductions in infusion mode was achieved with various modulation frequencies. The signal separation in the frequency region was successfully achieved for modulation frequencies up to 30 Hz with a sampling rate of 160 Hz.
We developed a point-of-care testing method based on a lateral flow assay for bevacizumab, using an anti-idiotypic DNA aptamer as the capture molecule. Bevacizumab loaded onto the sample pad on the assay strip bound to the red-colored aptamer-modified gold nanoparticles (AuNPs) in the conjugation pad and then migrated into the developing zone. The AuNPs bound to bevacizumab were trapped by protein A on the test spot. In contrast, excess amounts of unreacted AuNPs on the control spot, confirming that they migrated correctly. The assay strips enabled analysis within 20 min after adding the sample, and bevacizumab was quantified by photographing the colored areas with a smartphone and analyzing the images. The assay kit was capable of quantifying bevacizumab in the range of 0–200 μg/mL in Avastin diluent and bevacizumab-spiked human serum samples. This assay method enables on-site analysis of bevacizumab concentrations with a simple operation and without special analytical apparatus.
Amlodipine is a dihydropyridine drug that is used to lower high blood pressure. In this study, the photostabilities of orally disintegrating (OD) amlodipine tablets, both original and generic versions, and their powdered and suspended forms were evaluated. The concentration of the active pharmaceutical ingredient (API) was monitored by high-performance liquid chromatography (HPLC), revealing that residual amounts of API in powders and suspensions significantly decreased after ultraviolet light (UV) irradiation for 24 hr. Amlodipine OD tablets and their powders were packaged and stored at a house and a pharmacy for up to 90 days for the evaluation of their photostabilities in clinical use, and that amounts of API in the powdered form significantly decreased when they were stored in the house. ESI-LC/MS/MS analysis was performed to determine the chemical structure of photoproducts generated by photo-irradiated amlodipine OD tablets. It revealed that mainly a dehydrogenated form of amlodipine was generated concomitant with the photodegradation by UV and sunlight irradiation. This is the first study that evaluated the photostabilities of original amlodipine OD medicines, their generic OD versions and their altered forms.
Bendamustine (BD), a volatile anti-cancer drug in Japan, was quantitatively evaluated using solid phase extraction (SPE)-type extraction device designed for the adsorption of gaseous semi-volatile organic compounds. First, the stability of BD was tested in various conditions, including solvent, temperature, and contact with oxygen. Then, the BD standard solution was spiked on filter paper, and the SPE-type extraction device was connected to the filter downstream. The filter and extraction device were then used to collect clean air. The actual volatility of BD was determined in high-performance liquid chromatography. Even after 24 h of air sampling at 7 L/min (10,080 L), the results demonstrated that BD evaporation was not confirmed. Furthermore, BD evaporation was not confirmed in a 7,000 L air sampling volume of heated air at 50°C. The volatility properties of n-alkanes of C26H54, C28H58, and C30H62 in these air sampling conditions were compared. The results show that BD is not volatile at room temperature.