Recent progress in the extraction of terpenoids from essential oils and separation of the enantiomers by GC-MS.

Terpenoids possess significant physiological activities and are rich in essential oils. Some terpenoids have chiral centers and could form enantiomers with distinct physiological activities. Therefore, the extraction and separation of terpenoids enantiomers are very important and have attracted extensive attention in recent years. Meanwhile, the specific distribution and enantiomer excess results (the excess of one enantiomer over the other in a mixture of enantiomers) could be used as quality markers for illegitimate adulteration, origin identification, and exploring component variations and functional interrelations across different plant tissues. In this study, an overview of the progress in the extraction of terpenoids from essential oils and the separation of their enantiomers over the past two decades has been made. Extraction methods were retrieved by the resultant network visualization findings. The results showed that the predominant methods are hydrodistillation, solvent-free microwave extraction, headspace solid-phase microextraction and supercritical fluid extraction methods. GC-MS combined with chiral chromatography columns is commonly used for the separation of enantiomers, while 2D GC is found to have stronger resolution ability. Finally, some prospects for future research directions in the extraction and separation identification of essential oils are proposed.

UPLC-ESI-Q-TOF-MS/MS based metabolomics investigation on chemical constituent consistency of Zhenwu Decoction before and after compatibility.

Zhenwu Decoction (ZWD), a classic formula from Zhang Zhongjing's "Treatise on Typhoid Fever" in the Han Dynasty, consists of five traditional Chinese medicines: Aconiti Lateralis Radix Praeparata (ALRP), Paeoniae Radix Alba, Poria Cocos, Ginger, and Rhizoma Atractylodis Macrocephalae. To evaluate the chemical constituent consistency of ZWD before and after compatibility, an ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry was established to comprehensively study the constituents of ZWD. By normalizing the peak area, the pairwise compatibility of ALRP and the other four medicinal herbs, as well as the compatibility of the entire formula were studied, respectively. Multivariate statistical analysis was used to identify the differences. The processed data were analyzed by principal component analysis and supervised orthogonal partial least squared discriminant analysis, and an S-plot was generated to compare the differences in the chemical composition of the two types of decoction samples. The results showed that during the decoction process of ZWD, a total of seven components were recognized as differential compounds before and after compatibility of ZWD, namely 6-gingerol, zingerone, benzoylhypaconine, hypaconitine, benzoylaconine, paeoniflorin and fuziline. The results of this study provide basic data reference for understanding the law of ZWD compatibility and are valuable for the compatibility study of other herbal medicines.

A magnetic beads-based ligand fishing method Coupled with UHPLC-QTOF MS for screening and identification of α-glucosidase inhibitors from Houttuynia cordata Thunb.

In this study, a method for the screening and identification of α-glucosidase inhibitors from natural products was developed. The α-glucosidase was immobilized on carboxyl terminated magnetic beads to form a ligand fishing system to screen the potential inhibitors. A total of 9 compounds were fishing out from the crude Houttuynia cordata Thunb. extract. Meanwhile, ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) was used for the identification of the chemical structures, including 3 chlorogenic acid isomers, 2 flavone C-glycosides and 4 flavone O-glycosides. The combination of enzyme immobilization magnetic beads and UHPLC-QTOF MS could be used for the screening of bioactive multi-components from herbs with appropriate targets. Taking the advantage of the specificity of enzyme binding and the convenience of magnetic separation, the method has great potential for rapid screening of α-glucosidase inhibitors from complicated natural product extracts.

Differentiation of tetracyclines and their 4-epimers by mass spectrometry of the alkali metal adduct ions.

Tetracyclines (TCs) are a family of broad-spectrum antibiotics. During the manufacturing process or storage, epimerization of tetracyclines could occur, leading to 4-epimers which are nearly inactive. From an analytical point of view, isomers are often difficult to distinguish. Previously, four pairs of TCs (oxytetracycline, tetracycline, doxycycline, chlortetracycline and their respective 4-epimers) were differentiated by mass spectrometry (MS) through protonated ions. However, they do not follow common rules and so it is still quite difficult to differentiate between them. In order to solve this, the four pairs were differentiated in the current study by collision induced dissociation (CID) spectra of the alkali adduct ions, including lithium, sodium and potassium. In the spectra of the sodium adducts, all studied tetracyclines showed a tendency to form [M+Na-NH3]+ ions, while the 4-epimers liked to form [M+Na-NH3-H2O]+ ions. Meanwhile, energy resolved mass spectrometry (ERMS) showed that all four 4-epimers' sodium adducts had the tendency to fragment at higher energy points. In the CID spectra of lithium adducts of TCs, a similar trend was observed for three pairs, except for doxycycline. For potassium adducts, the fragmentation was found to be less discriminative. As was derived from the 3D model, the four pairs all interact with the alkali metal through the dimethyl amino group at the C-4 position. The lithium adduct species also bound through the hydroxyl group at the C-5 position. If the TCs did not have a hydroxyl group at the C-5 position, they bound with the hydroxyl group at the C-6 position. For the same TC, with an increase of the diameter of the metal ion, the loss of H2O decreased gradually. As sodium adduct ions are common during the ionization process, TCs and their 4-epimers could be differentiated rapidly by ERMS of the sodium adduct ions.

A high-throughput fully automatic biosensing platform for efficient COVID-19 detection.

We propose a label-free biosensor based on a porous silicon resonant microcavity and localized surface plasmon resonance. The biosensor detects SARS-CoV-2 antigen based on engineered trimeric angiotensin converting enzyme-2 binding protein, which is conserved across different variants. Robotic arms run the detection process including sample loading, incubation, sensor surface rinsing, and optical measurements using a portable spectrometer. Both the biosensor and the optical measurement system are readily scalable to accommodate testing a wide range of sample numbers. The limit of detection is 100 TCID50/ml. The detection time is 5 min, and the throughput of one single robotic site is up to 384 specimens in 30 min. The measurement interface requires little training, has standard operation, and therefore is suitable for widespread use in rapid and onsite COVID-19 screening or surveillance.

Deep learning in automatic detection of dysphonia: Comparing acoustic features and developing a generalizable framework.

BACKGROUND: Auditory-perceptual assessment of voice is a subjective procedure. Artificial intelligence with deep learning (DL) may improve the consistency and accessibility of this task. It is unclear how a DL model performs on different acoustic features. AIMS: To develop a generalizable DL framework for identifying dysphonia using a multidimensional acoustic feature. METHODS & PROCEDURES: Recordings of sustained phonations of /a/ and /i/ were retrospectively collected from a clinical database. Subjects contained 238 dysphonic and 223 vocally healthy speakers of Chinese Mandarin. All audio clips were split into multiple 1.5-s segments and normalized to the same loudness level. Mel frequency cepstral coefficients and mel-spectrogram were extracted from these standardized segments. Each set of features was used in a convolutional neural network (CNN) to perform a binary classification task. The best feature was obtained through a five-fold cross-validation on a random selection of 80% data. The resultant DL framework was tested on the remaining 20% data and a public German voice database. The performance of the DL framework was compared with those of two baseline machine-learning models. OUTCOMES & RESULTS: The mel-spectrogram yielded the best model performance, with a mean area under the receiver operating characteristic curve of 0.972 and an accuracy of 92% in classifying audio segments. The resultant DL framework significantly outperformed both baseline models in detecting dysphonic subjects on both test sets. The best outcomes were achieved when classifications were made based on all segments of both vowels, with 95% accuracy, 92% recall, 98% precision and 98% specificity on the Chinese test set, and 92%, 95%, 90% and 89%, respectively, on the German set. CONCLUSIONS & IMPLICATIONS: This study demonstrates the feasibility of DL for automatic detection of dysphonia. The mel-spectrogram is a preferred acoustic feature for the task. This framework may be used for vocal health screening and facilitate automatic perceptual evaluation of voice in the era of big data. WHAT THIS PAPER ADDS: What is already known on this subject Auditory-perceptual assessment is the current gold standard in clinical evaluation of voice quality, but its value may be limited by the rater's reliability and accessibility. DL is a new method of artificial intelligence that can overcome these disadvantages and promote automatic voice assessment. This study explored the feasibility of a DL approach for automatic detection of dysphonia, along with a quantitative comparison of two common sets of acoustic features. What this study adds to existing knowledge A CNN model is excellent at decoding multidimensional acoustic features, outperforming the baseline parameter-based models in identifying dysphonic voices. The first 13 mel-frequency cepstral coefficients (MFCCs) are sufficient for this task. The mel-spectrogram results in greater performance, indicating the acoustic features are presented in a more favourable way than the MFCCs to the CNN model. What are the potential or actual clinical implications of this work? DL is a feasible method for the detection of dysphonia. The current DL framework may be used for remote vocal health screening or documenting voice recovery after treatment. In future, DL models may potentially be used to perform auditory-perceptual tasks in an automatic, efficient, reliable and low-cost manner.

Study of Chemical Compositions and Anticancer Effects of Paris polyphylla var. Chinensis Leaves.

Paris polyphylla var. chinensis (Franch.) Hara is a perennial herb belonging to the Trilliaceae family. Ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to detect the composition of different fractions of Paris polyphylla var. chinensis leaves. Meanwhile, the extracts of different fractions were evaluated for their cytotoxic activities against four selected human cancer cell lines and one human normal epithelial cell line based on the MTT assay method. Multivariate statistical analysis was performed to screen differential compounds and to analyze the distributions between different fractions. Finally, more than 60 compounds were obtained and identified from the different fractions of Paris polyphylla var. chinensis leaves, and the chloroform and n-butanol extracts showed significant cytotoxic effects on these four cancer cells. Several compounds were preliminarily identified from different fractions, including 36 steroidal saponins, 11 flavonoids, 10 ceramides, 8 lipids, 6 organic acids, and 8 other compounds. Various compounds were screened out as different chemical components of different fractions, which were considered as a potential substance basis for the cytotoxicity of Paris polyphylla var. chinensis leaves.

Rapid Optical Biosensing of SARS-CoV-2 Spike Proteins in Artificial Samples.

Tests for SARS-CoV-2 are crucial for the mass surveillance of the incidence of infection. The long waiting time for classic nucleic acid test results highlights the importance of developing alternative rapid biosensing methods. Herein, we propose a fiber-optic biolayer interferometry-based biosensor (FO-BLI) to detect SARS-CoV-2 spike proteins, extracellular domain (ECD), and receptor-binding domain (RBD) in artificial samples in 13 min. The FO-BLI biosensor utilized an antibody pair to capture and detect the spike proteins. The secondary antibody conjugated with horseradish peroxidase (HRP) reacted with the enzyme substrate for signal amplification. Two types of substrates, 3,3'-diaminobenzidine (DAB) and an advanced 3-Amino-9-ethylcarbazole (i.e., AMEC), were applied to evaluate their capabilities in enhancing signals and reaching high sensitivity. After careful comparison, the AMEC-based FO-BLI biosensor showed better assay performance, which detected ECD at a concentration of 32-720 pM and RBD of 12.5-400 pM in artificial saliva and serum, respectively. The limit of detection (LoD) for SARS-CoV-2 ECD and RBD was defined to be 36 pM and 12.5 pM, respectively. Morphology of the metal precipitates generated by the AMEC-HRP reaction in the fiber tips was observed using field emission scanning electron microscopy (SEM). Collectively, the developed FO-BLI biosensor has the potential to rapidly detect SARS-CoV-2 antigens and provide guidance for "sample-collect and result-out on-site" mode.

Diastereomer recognition of three pairs of tetracyclines by electrospray ionization mass spectrometry.

RATIONALE: Stereoisomer profiling is always a difficult issue. Based on the difference between diastereomers, usually because of steric hindrance, isomers can be differentiated by mass spectrometry (MS), although it is often not an easy task. In the current study, tetracycline, chlortetracycline and doxycycline could be distinguished from their respective 4-epimers by MS. METHODS: The electrospray ionization tandem mass spectrometry (ESI-MSn ) analyses were carried out on a Bruker 3000plus ion trap mass spectrometer. For MS/MS experiments, the collision energy was set between 0.18 and 0.45 V to perform energy-resolved mass spectrometry (ERMS). Test solutions were prepared in methanol/water (90:10, v/v) at a concentration of 10 µg/mL. RESULTS: Compared with the collision-induced dissociation (CID) spectrum of protonated tetracycline, the most abundant peak changed from m/z 427 to m/z 410 for 4-epitetracycline. For chlortetracycline and its 4-epimer, differences in relative abundance were observed too. In the CID spectrum of a fragment ion of doxycycline, the abundance of m/z 154 was relatively higher than for the 4-epimer, showing the same trend as in the CID spectra of the other two pairs of tetracyclines. CONCLUSIONS: The CID spectra of tetracycline and chlortetracycline were different from those of their 4-epimers. The CID spectra of protonated doxycycline and its 4-epimer showed only a subtle difference, but the m/z 154 fragment ion in the CID spectra of the fragment ion at m/z 428 offers the possibility to differentiate both epimers.

On-Site Biolayer Interferometry-Based Biosensing of Carbamazepine in Whole Blood of Epileptic Patients.

On-site monitoring of carbamazepine (CBZ) that allows rapid, sensitive, automatic, and high-throughput detection directly from whole blood is of urgent demand in current clinical practice for precision medicine. Herein, we developed two types (being indirect vs. direct) of fiber-optic biolayer interferometry (FO-BLI) biosensors for on-site CBZ monitoring. The indirect FO-BLI biosensor preincubated samples with monoclonal antibodies towards CBZ (MA-CBZ), and the mixture competes with immobilized CBZ to bind towards MA-CBZ. The direct FO-BLI biosensor used sample CBZ and CBZ-horseradish peroxidase (CBZ-HRP) conjugate to directly compete for binding with immobilized MA-CBZ, followed by a metal precipitate 3,3'-diaminobenzidine to amplify the signals. Indirect FO-BLI detected CBZ within its therapeutic range and was regenerated up to 12 times with negligible baseline drift, but reported results in 25 min. However, Direct FO-BLI achieved CBZ detection in approximately 7.5 min, down to as low as 10 ng/mL, with good accuracy, specificity and negligible matric interference using a high-salt buffer. Validation of Direct FO-BLI using six paired sera and whole blood from epileptic patients showed excellent agreement with ultra-performance liquid chromatography. Being automated and able to achieve high throughput, Direct FO-BLI proved itself to be more effective for integration into the clinic by delivering CBZ values from whole blood within minutes.

Components identification and isomers differentiation in pigeon pea (Cajanus cajan L.) leaves by LC-MS.

A valid and reliable method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry using electrospray ionization was established to identify chemical constituents in the ethanol extract of pigeon pea leaves. A total of 58 compounds were detected both in positive and negative modes. Among them, 42 compounds, including 16 flavones, 1 flavonol, 5 flavanones, 9 isoflavones, 1 coumarin, 1 lactone, 6 stilbenes, 2 chalcones, and 1 other compound, were unambiguously identified or tentatively assigned in view of the retention time, the molecular formula, as well as the fragmentation patterns. Moreover, eight sets of isomers were differentiated by the ion trap mass spectrometry based on the fragment ion differences or the abundance differences of the same fragment ions. The energy-resolved mass spectrometry in light of the relative abundance of characteristic fragment ions was adopted in the study.

Fragmentation pathways of deprotonated amide-sulfonamide CXCR4 inhibitors investigated by ESI-IT-MSn , ESI-Q-TOF-MS/MS and DFT calculations.

Amide-sulfonamides provide a potent anti-inflammatory scaffold targeting the CXCR4 receptor. A series of novel amide-sulfonamide derivatives were investigated for their gas-phase fragmentation behaviors using electrospray ionization ion trap mass spectrometry and quadrupole time-of-flight mass spectrometry in negative ion mode. Upon collision-induced dissociation (CID), deprotonated amide-sulfonamides mainly underwent either an elimination of the amine to form the sulfonyl anion and amide anion or a benzoylamide derivative to provide sulfonamide anion bearing respective substituent groups. Based on the characteristic fragment ions and the deuterium-hydrogen exchange experiments, three possible fragmentation mechanisms corresponding to ion-neutral complexes including [sulfonyl anion/amine] complex (INC-1), [sulfonamide anion/benzoylamide derivative] complex (INC-2) and [amide anion/sulfonamide] complex (INC-3), respectively, were proposed. These three ion-neutral complexes might be produced by the cleavages of S-N and C-N bond from the amide-sulfonamides, which generated the sulfonyl anion (Route 1), sulfonamide anion (Route 2) and the amide anion (Route 3). DFT calculations suggested that Route 1, which generated the sulfonyl anion (ion c) is more favorable. In addition, the elimination of SO2 through a three-membered-ring transition state followed by the formation of C-N was observed for all the amide-sulfonamides.

Simultaneous Quantification and Pharmacokinetic Study of Nine Bioactive Components of Orthosiphon stamineus Benth. Extract in Rat Plasma by UHPLC-MS/MS.

Orthosiphon stamineus Benth. (OS) is a traditional folk medicine for the treatment of kidney stones and other urinary tract diseases. In this study, a rapid and sensitive Ultra high-performance liquid chromatography (UHPLC)-MS/MS approach was established and validated for the simultaneous quantification of nine bioactive components in rat plasma. The nine components from OS extract detected in rat plasma were danshensu, protocatechuic acid, caffeic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, cichoric acid, sinensetin and eupatorin. After liquid-liquid extraction with ethyl acetate, the plasma samples were subjected to a triple quadrupole mass spectrometer employing electrospray ionization (ESI) technique and operating in multiple reaction monitoring (MRM) with both positive and negative ion modes. The standard curves showed good linear regression (r > 0.9915) over the concentration range for the nine analytes. The inter-day and intra-day precision and accuracy were found to be within 15% of the nominal concentration. The recovery and stability of nine compounds were all demonstrated to be within acceptable limits. The approach was successfully applied to investigate the pharmacokinetic analysis of the nine bioactive components after oral administration of OS extract in rats.

Separation and characterization of unknown impurities in latamoxef sodium by LC-Q-TOF MS and a summary of their positive-ion fragmentation regularities.

A simple and sensitive method was developed for separation and characterization of seventeen impurities from commercial latamoxef sodium for injection by liquid chromatography combined with electrospray ionization and QTOF mass spectrometer (LC-ESI-QTOF MS). The chromatographic separation was performed on a Boston Green ODS-AQ C18 column (250 mm × 4.6 mm, 5 µm) under gradient mode using binary mobile phase: (A) ammonium acetate (10 mM)-methanol (99:1, v/v) and (B) ammonium acetate (10 mM)-methanol (70:30, v/v). Based on tandem multistage MS and high resolution MS data, the molecular formulas and structures of unknown impurities were inferred. A plausible formation mechanism of impurities was also proposed. In addition, the fragmentation regularity of degraded impurities in positive-ion mode was summarized.

Development of a reversed phase liquid chromatographic method for analysis of pyridoxal-5'-phosphate and its impurities.

Since no proper method is available in literature for the analysis of pyridoxal-5'-phosphate, a reversed phase liquid chromatographic method was developed and validated for specificity, sensitivity, linearity, precision and accuracy. Nine potential related substances and forced degradation products could be successfully separated from the main peak. The separation was achieved on a Polaris C18 column (250 × 4.6 mm i.d., 5 µm) using a mobile phase consisting of 20 mM ammonium formate in 0.65% formic acid - acetonitrile (98.8:1.2, v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min and the analytes were detected by UV at 240 nm. The volatile mobile phase allowed also direct coupling to an ion-trap mass spectrometer with a positive electrospray ionization source to characterize unknown peaks in the chromatogram. The method can be used for quality control purposes as required by regulatory authorities to ensure the product's safety and efficacy.

Improved liquid chromatographic method for quality control of spiramycin using superficially porous particles.

This article describes the development and validation of a liquid chromatographic method for spiramycin using a column with superficially porous particles. Gradient elution was applied and the mobile phase consisted of phosphate buffer (0.2M; pH 8.3) - H2O - acetonitrile in a ratio 10:60:30 (v/v/v) for mobile phase A and 10:30:60 (v/v/v) for mobile phase B. UV detection was performed at 232nm. Compared to previous methods, the analysis time was about two times faster and impurities were better separated. Furthermore, impurities which were present above 0.25% were characterized using liquid chromatography coupled with mass spectrometry (LC/MS).

Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-ESI-ion trap MS and LC-ESI-QTOF MS.

As requested by regulatory authorities, impurity profiling is an important issue of quality control. In this work, a simple and sensitive liquid chromatographic (LC) method compatible with mass spectrometry (MS) was developed to study related substances and degradation products in sodium cromoglycate drug substance and eye drops. The method used a Sunfire column (4.6mm×150mm, 3.5µm). Mobile phase A consisted of 10mM ammonium formate and mobile phase B was acetonitrile. Linear gradient elution with a post-run time of 8min was performed as follows: 0-30min, 3% B to 50% B; 30-35min, 50% B. The flow rate was set at 1.0mL/min. Degradation experiments were performed to check the stability indicating properties of the developed method. Based on MSn spectral data and exact mass measurements, the chemical structures of 2 unknown impurities and 6 unknown degradation products were characterized, including impurity C listed in the European Pharmacopoeia as unknown structure. In addition, a plausible mechanism for the formation of the degradation products was also proposed.

Development and validation of a liquid chromatographic method for the analysis of squaric acid dibutyl ester and its impurities.

A simple, fast and selective stability indicating liquid chromatographic method has been described for the simultaneous determination of squaric acid dibutyl ester and its impurities. The chromatographic separation was achieved on a C2 column (250mm×4.6mm i.d., 5µm) using a mobile phase consisting of 0.15% phosphoric acid - acetonitrile - methanol (30:60:10, v/v/v). Isocratic elution was performed at a flow rate of 1.0mLmin-1. The analytes were detected by UV at 252nm. The method was validated according to the ICH guidelines and satisfactory results were obtained. The specificity of the developed method was tested using forced degradation solutions of the drug substance. Characterization of squaric acid dibutyl ester and its forced degradation products was achieved by coupling mass spectrometry (MS) to the liquid chromatographic (LC) system. The method was successfully applied for quality control purposes including assay and determination of related compounds as required by regulatory guidelines to ensure its safety and efficacy since no monograph is available in official compendia.

Characterization of an unknown impurity in doxofylline using LC-MS and NMR.

During quality control of doxofylline, a novel impurity was detected, which was above the identification threshold defined by ICH. First, a liquid chromatographic method compatible with mass spectrometric (MS) detection was developed. Based on tandem multistage MS and high resolution MS data, the unknown impurity was found to consist of two theophylline groups connected by a methylene group. The structure was further confirmed by 1D and 2D nuclear magnetic resonance (NMR) experiments after semi-preparative isolation. In addition, the formation of the impurity was also discussed.