Traditional Chromatographic Methods Research Articles

A fully automatic system for underway N2O measurements based on cavity ring-down spectroscopy

ABSTRACTN2O is one of the most important greenhouse and ozone-depleting gases and has been the source of considerable concern in recent years. The oceans account for ~ 1/4 of the global N2O emission budget; however, the oceanic N2O source/sink characteristics are not well understood. To enhance the study of oceanic N2O source/sink characteristics, our laboratory developed a fully automatic underway system for surface water N2O concentration and atmospheric N2O mole fraction measurements consisting of a cavity ring-down spectroscopy (CRDS) instrument and an upstream device. The developed device can be programmed to switch the CRDS measurements from the equilibrator headspace to the atmospheric sample and the reference gas sample. The surface water N2O concentration is calculated from the equilibrium headspace N2O mole fraction in the equilibrator. The response time of this equilibrator is ~ 3.4 min, and the estimated precision of this method for surface water N2O measurements is better than 0.5% (relative standard deviation, RSD), which is one order of magnitude better than that of traditional gas chromatographic methods and can be further optimised. Data are acquired every 20 s, and the calibration frequency requirement of this system is approximately 7–10 days. This labor-saving underway system is a powerful tool for high-precision and high-resolution measurements of atmospheric and oceanic N2O and can significantly improve the study of the characteristics of oceanic N2O sources/sinks and their response to climate change.

Enzyme-Based Biosensors for Electrochemical Detection of Pesticides–A Mini Review

Despite their important contribution in increasing crops production, most pesticides are harmful to humans and living beings and can persist in the environment over long a long duration. Traditional chromatographic methods of analysis are expensive and cumbersome. Biosensor technology appears therefore as an efficient and economical alternative for fast detection of pesticides. The devices are portable, rapid, and highly sensitive. Other important features of the devices are their relatively high sensistivity and low response time. Enzymatic biosensors for pesticide detection rely either on the inhibition mechanism or on the catalytic activity of the immobilized enzyme toward a specific pesticide. Metal and carbon based nanomaterials are being widely used as immobilization support owing to novel characteristics such as biocompatibility and enhanced electron transfer ability for sensitive electrochemical detection, among others. This review focusses on the electrochemical detection of organophosphorus pesticides, delineating the limit of detection and response time of biosensors toward a wide range of organophosphorus pesticides.

Enzyme-Based Biosensors for Electrochemical Detection of Pesticides–A Mini Review

Despite their important contribution in increasing crops production, most pesticides are harmful to humans and living beings and can persist in the environment over long a long duration. Traditional chromatographic methods of analysis are expensive and cumbersome. Biosensor technology appears therefore as an efficient and economical alternative for fast detection of pesticides. The devices are portable, rapid, and highly sensitive. Other important features of the devices are their relatively high sensistivity and low response time. Enzymatic biosensors for pesticide detection rely either on the inhibition mechanism or on the catalytic activity of the immobilized enzyme toward a specific pesticide. Metal and carbon based nanomaterials are being widely used as immobilization support owing to novel characteristics such as biocompatibility and enhanced electron transfer ability for sensitive electrochemical detection, among others. This review focusses on the electrochemical detection of organophosphorus pesticides, delineating the limit of detection and response time of biosensors toward a wide range of organophosphorus pesticides.

Solubilization, Folding, and Purification of a Recombinant Peptidoglycan-Associated Lipoprotein (PAL) Expressed in Escherichia coli.

Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan-associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram-negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production. This protocol describes an efficient protein refolding method to solubilize and purify a recombinant PAL. Initially, recombinant PAL-enriched inclusion bodies obtained after the induction of PAL expression in Escherichia coli are treated with 8 M urea and then undergo buffer exchange via dialysis. Afterward, the soluble, recombinant PAL is purified using standard chromatographic methods. © 2018 by John Wiley & Sons, Inc.

Quantitation of Saccharin and Cyclamate in Tabletop Formulations by Portable Raman and NIR Spectrometers in Combination with Partial Least Squares Regression

Rapid, direct, and reagent-free screening tools using vibrational spectroscopy in combination with partial least squares regression (PLSR) were developed for the determination of sodium saccharin and sodium cyclamate in tabletop formulations. The four vibrational spectroscopic instruments employed were a portable Raman spectrometer, a NIR handheld device, and two FT-NIR benchtop spectrometers. Wavenumber ranges and type of spectral pretreatment were optimized for each PLSR calibration model using an independent validation set. Each sweetener model provided reliable predictions (low errors in validation and r 2 above 0.90) for both saccharin and cyclamate samples. Optimized models were tested with four commercially available tabletop formulations in order to simulate the application of the developed models towards routine sweetener analysis. With the exception of one model, the sweetener concentration predictions in commercial tabletop formulations using the portable devices were not significantly different from those based on spectra collected on the benchtop spectrometers. PLSR-predicted mean sweetener concentrations were within 80–120% of the label declared values, while the sweetener without a label declaration had consistent concentrations across the analytical methods used. As shown by the good agreement between spectroscopic and chromatographic analyses, the portable spectrometers offer an alternative to traditional chromatographic methods. To our knowledge, this is the first time portable Raman and handheld NIR devices with PLSR calibration models have been employed to evaluate sweeteners, and these analytical methods hold potential to be used for rapid screening of tabletop formulations for quality assurance and for regulatory labeling verification.

A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways.

The implementation of mass spectrometry (MS)-based metabolomics is advancing many areas of biomedical research. The time associated with traditional chromatographic methods for resolving metabolites prior to mass analysis has limited the potential to perform large-scale, highly powered metabolomics studies and clinical applications. Here we describe a three-minute method for the rapid profiling of central metabolic pathways through UHPLC/MS, tracing experiments in vitro and in vivo, and targeted quantification of compounds of interest using spiked in heavy labeled standards. This method has shown to be linear, reproducible, selective, sensitive, and robust for the semi-targeted analysis of central carbon and nitrogen metabolism. Isotopically labeled internal standards are used for absolute quantitation of steady-state metabolite levels and de novo synthesized metabolites in tracing studies. We further propose exploratory applications to biofluids, cell and tissue extracts derived from relevant biomedical/clinical samples. While limited to the analysis of central carbon and nitrogen metabolism, this method enables the analysis of hundreds of samples per day derived from diverse biological matrices. This approach makes it possible to analyze samples from large patient populations for translational research, personalized medicine, and clinical metabolomics applications. Copyright © 2017 John Wiley & Sons, Ltd.

Alternating isocratic and step gradient elution high-speed counter-current chromatography for the isolation of minor phenolics from Ormocarpum kirkii bark

A total of 14 compounds were isolated from the ethanol bark extract of O. kirkii S. Moore (Fabaceae) by alternating isocratic and step gradient elution high-speed counter-current chromatography (HSCCC) methods, using several solvent systems with reference to the polarity of compounds being purified. The extract was successively fractionated with generic solvent systems including n-hexane-ethanol-water (4:2:2) and ethyl acetate-water (1:1). Resulting fractions were further purified using the following preparative gradient elution consisting of ethyl acetate-n-butanol-water (X:Y:10), (X:Y=9:1 (I); 8:2 (II); 7:3 (III); 6:4 (IV); 5:5 (V); 4:6 (VI) 3:7 (VII) and n-hexane- ethyl acetate-methanol-water (1:X:1:1), X=1, 2, 2.5, 3 solvent systems. Two flavone glycosides, apigenin-6-C-β-d-glucopyranosyl-4′-O-[β-d-glucopyranosyl-(1→5)]-β-d-apiofuranoside (1) and apigenin-6-C-β-d-glucopyranosyl-4′-O-β-d-apiofuranoside (2), and one biflavanone diglycoside 7,7″-di-O-β-d-glucosylliquiritigeninyl-(I-3,II-3)-naringenin (4) were isolated as new compounds along with other 11 known ones. The structures of the isolated compounds were identified by HPLC-UV, ESI-MS, 1D and 2D NMR and comparison with literature data. Thus, over common traditional chromatographic methods, the present study shows that HSCCC is a useful and fast method for natural product research with no losses and lower solvent use.

Micellar liquid chromatographic determination of metformin hydrochloride using fluorimetric detection after pre-column derivatization: application to pharmacokinetic parameters in immediate and sustained release formulations.

An accurate and selective method using micellar liquid chromatography was developed to determine metformin hydrochloride both in its pharmaceutical dosage forms and human plasma. Separation was conducted using a Zorbax SB-Phenyl (250×4.6mm id) stainless steel column at ambient temperature after pre-column derivatization with 9,10-phenanthraquinone. A mobile phase composed of 0.1M sodium dodecyl sulfate, 10% 1-propanol and triethylamine (0.3%) in 0.02M phosphoric acid, adjusted to pH 2.5, was used at a flow rate of 1ml/min with fluorimetric detection at 450nm after excitation at 306nm. The proposed method showed high sensitivity with limit of quantification of 0.35μg/ml and limit of detection of 0.23μg/ml, being linear from 0.5 to 3.0μg/ml. Being highly sensitive, the method could be applied to spiked human plasma, and also to follow the pharmacokinetic parameters of the studied drug in healthy volunteers after administration of both its immediate and sustained release tablet formulations. Such procedures were carried out without any extraction steps, which improves the accuracy and precision of the proposed method when applied to human plasma. Detailed validation procedures were also carried out giving results in accordance with the comparison method. The proposed method has also the advantage of being environmentally safe, where the use of organic solvents is highly limited in comparison with other traditional chromatographic separation methods that depend mainly on a high proportion of organic modifiers. This concept, in turn, emphasizes the application of green chemistry in the analysis of pharmaceutical products. The simplicity, relatively low cost and short analysis time of the suggested method makes it a candidate for routine quality control work.

Ultra-high performance supercritical fluid chromatography of lignin-derived phenols from alkaline cupric oxide oxidation.

Traditional chromatographic methods for the analysis of lignin‐derived phenolic compounds in environmental samples are generally time consuming. In this work, an ultra‐high performance supercritical fluid chromatography method with a diode array detector for the analysis of major lignin‐derived phenolic compounds produced by alkaline cupric oxide oxidation was developed. In an analysis of a collection of 11 representative monomeric lignin phenolic compounds, all compounds were clearly separated within 6 min with excellent peak shapes, with a limit of detection of 0.5–2.5 μM, a limit of quantification of 2.5–5.0 μM, and a dynamic range of 5.0–2.0 mM (R 2 > 0.997). The new ultra‐high performance supercritical fluid chromatography method was also applied for the qualitative and quantitative analysis of lignin‐derived phenolic compounds obtained upon alkaline cupric oxide oxidation of a commercial humic acid. Ten out of the previous eleven model compounds could be quantified in the oxidized humic acid sample. The high separation power and short analysis time obtained demonstrate for the first time that supercritical fluid chromatography is a fast and reliable technique for the analysis of lignin‐derived phenols in complex environmental samples.

Discriminating Lamiophlomis rotata According to Geographical Origin by (1)H-NMR Spectroscopy and Multivariate Analysis.

Lamiophlomis rotata (Duyiwei) is a folk herbal medicine that traditionally has been used in China as a hemostatic agent. Raw plant materials used for medicinal products from different geographical regions are often inconsistent in chemical composition. Metabolic fingerprinting provides a new approach for distinguishing the geographical origins of L. rotata. To identify metabolites that contribute to the different geographical regions of L. rotata samples. Lamiophlomis rotata metabolomics were performed by (1)H-nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analyses. The L. rotata metabolic profile was prepared for NMR measurements using methanol-d4 solvent. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to analyse the L. rotata (1)H-NMR spectroscopy data. Nine iridoid glycosides, one flavonoid and three phenylpropanoid glycosides were detected in L. rotata by (1)H-NMR spectroscopy. (1)H-NMR measurements and multivariate analysis were used to successfully discriminate samples from three different locations. The NMR-based analysis of L. rotata is a more comprehensive approach than traditional chromatographic methods. Simple sample preparation, rapidity and reproducibility of are additional advantages of NMR analysis.

Microchip electrophoresis for fast and interference-free determination of trace amounts of glyphosate and glufosinate residues in agricultural products.

Fast screening of herbicide residues is becoming important to ensure food safety, but traditional chromatographic methods may not be suitable for rapid on-site analysis of samples with complicated matrices. Here, we describe a method for rapid and sensitive determination of glyphosate (GLYP) and glufosinate (GLUF) residues in agricultural products by electrophoresis on disposable microchips with laser-induced fluorescence detection. With this method, quantitative analysis of trace amounts of GLYP and GLUF can be achieved with relatively simple sample preparation.

Kaizen Approach to Evaluate Different Oil Sample & Compare with Traditional Chromatographic Approach

Oil is one of the important formulations in Ayurveda for external as well as internal use. In this study, an evaluation of the commercially available Mahamasa, Mahanarayana, sesamum oil was conducted using two different chromatographic Technique (traditional & modified pre chromatographic TLC).HPTLC is an important designing tool to evaluate the oil sample .But sampling strategy of oil in the context of traditional chromatographic method is very lengthy, costly and less convenient. Here we have modulate the chromatographic method with respect to continuous improvement plan(Kaizen approach) such as need based acceptation of saponification and paradigm shift of direct application of oil on TLC plate. Direct application of oil on plate concept adopted from pre-chromatographic derivatization, which oil sample direct applied on TLC plate and developed with suitable solvent system, and during development only unsap material will we separate on TLC plate and then compare with traditional method outcome. Separation pattern of oil on plate are same both method traditional and modified method. That means separate operation will not require for oil separation like hydrolysis of oil, extraction of unsaponifiable material and sample preparation. However modified method is less time, chemical consuming and more convenient for economically chromatographic evaluation of oil sample.

Diagnostic challenges of aminoacidopathies and organic acidemias in a developing country: A twelve-year experience

BackgroundDiagnosis of aminoacidopathies and organic acidemias constitutes a real challenge in a developing country with high consanguinity rate and no systematic newborn screening. We report a twelve-year experience with the identification of these disorders in Lebanon, based on their clinical and biochemical profiles. MethodsIn this retrospective study, we reviewed clinical presentation and biochemical investigations of 294 patients. Traditional chromatographic methods were used for analyses. Findings were linked to the identified disorders. ResultsOut of 2921 patients, presenting to our metabolic program with neurological, digestive, family history and/or other symptoms suggestive of aminoacidopathy or organic acidemia, 294 patients were included with confirmed amino or organic acid disorder. The overall analytical yield was 10%. Aminoacidopathies were three-fold higher than organic acidemias. Phenylketonuria and methylmalonic acidemia were the most frequent. The majority of patients (79%) were symptomatic (median age: 14months, range: 1day–44years), mainly with neurological manifestations (87%). Intellectual disability was mostly due to phenylketonuria (73%). Chronic liver failure was frequent in maple syrup urine disease (53%). Plasma amino and urine organic acid chromatography were diagnostic in 8.8% and 3.9% of analyzed cases, respectively. Change in chromatographic technique from reversed-phase to ion-exchange enhanced the detection of many aminoacidopathies. ConclusionsIn the absence of newborn screening, the majority of aminoacidopathy and organic acidemia cases are still diagnosed clinically. This study emphasizes the importance of clinical awareness and accurate biochemical analyses as key tools for diagnosis in countries like ours, and the necessity for a comprehensive national newborn screening program.

Paper Analytical Devices for Fast Field Screening of Beta Lactam Antibiotics and Antituberculosis Pharmaceuticals

Reports of low-quality pharmaceuticals have been on the rise in the past decade, with the greatest prevalence of substandard medicines in developing countries, where lapses in manufacturing quality control or breaches in the supply chain allow substandard medicines to reach the marketplace. Here, we describe inexpensive test cards for fast field screening of pharmaceutical dosage forms containing beta lactam antibiotics or combinations of the four first-line antituberculosis (TB) drugs. The devices detect the active pharmaceutical ingredients (APIs) ampicillin, amoxicillin, rifampicin, isoniazid, ethambutol, and pyrazinamide and also screen for substitute pharmaceuticals, such as acetaminophen and chloroquine that may be found in counterfeit pharmaceuticals. The tests can detect binders and fillers such as chalk, talc, and starch not revealed by traditional chromatographic methods. These paper devices contain 12 lanes, separated by hydrophobic barriers, with different reagents deposited in the lanes. The user rubs some of the solid pharmaceutical across the lanes and dips the edge of the paper into water. As water climbs up the lanes by capillary action, it triggers a library of different chemical tests and a timer to indicate when the tests are completed. The reactions in each lane generate colors to form a "color bar code" which can be analyzed visually by comparison with standard outcomes. Although quantification of the APIs is poor compared with conventional analytical methods, the sensitivity and selectivity for the analytes is high enough to pick out suspicious formulations containing no API or a substitute API as well as formulations containing APIs that have been "cut" with inactive ingredients.

Purification and characterization of a 1,3-β-D-glucan recognition protein from Antheraea pernyi larve that is regulated after a specific immune challenge

Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-β-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-βGRP). Ap-βGRP was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-βGRP specifically bind 1,3-β-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-β-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-βGRP antibody could be recovered by addition of purified Ap-βGRP. These results demonstrate that Ap-βGRP acts as a biosensor of 1,3-β-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-β-D-glucan or Ap-βGRP alone was unable to trigger the proPO system, but they both did. Ap-βGRP was specifically degraded following the activation of proPO with 1,3-β-Dglucan. These results indicate the variation in the amount of Ap-βGRP after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response. [BMB Reports 2013; 46(5): 264-269]

Rapid Determination of Chloramphenicol in Honey by Laser Diode Thermal Desorption Using Atmospheric Pressure Chemical Ionization-Tandem Mass Spectrometry

Abstract A high-throughput, rapid, and reliable method based on laser diode thermal desorption (LDTD) and atmospheric pressure chemical ionization coupled to tandem MS (APCI-MS/MS) was used to identify and quantify chloramphenicol (CAP) residues in honey. Sample pretreatment consisted of a liquid–liquid extraction of diluted honey in water with ethyl acetate containing stearic acid. After extraction, a 2 μL aliquot of the organic phase was deposited into a 96-well plate prior to detection by LDTD-APCI-MS/MS. The total analysis time was less than 6 s compared to several minutes for traditional chromatographic methods used for CAP detection, since no chromatographic separation was necessary. The extraction and analysis were made with honey samples of different color grading ranging from extra white to dark amber. The method showed good linearity (R2 = 0.99995) within a concentration range of 0.1 to 500 ng/g for CAP with D5-CAP as an internal standard. The RSD varied between 8 and 24% over the calibration range (n = 4). The method LOD for CAP in honey was 0.19 ng/g. This work demonstrates that LDTD-APCI-MS/MS could be used for fast and effective quantification of CAP in honey samples.

Fast HPLC-DAD quantification procedure for selected sulfonamids, metronidazole and chloramphenicol in wastewaters using second-order calibration based on MCR-ALS

The present work focuses on the application of multivariate curve resolution-alternating least squares (MCR-ALS) for analysis of five important antibiotics (sulfamethoxazole, metronidazole, chloramphenicol, sulfadizine and sulfamerazine) in highly complex wastewater samples, using solid phase extraction (SPE)-high performance liquid chromatography with diode-array detection (HPLC-DAD) on a short column, regarding the fast elution methodology. The samples were pre-concentrated on Bond Elut-ENV cartridges and separated on an ODS column (7cm) in less than 4min using an isocratic mode of elution with methanol–water (55:45, v/v) at pH=3.2. Due to the matrix interferences and the resulting sensitivity changes, a strategy implementing standard addition calibration in combination with MCR/ALS algorithm was applied. In this paper, the signal corresponding to background contribution was considered as a systematic part of the model during MCR/ALS data processing, so the background correction step was not necessary. The qualitative and quantitative results showed that the application of MCR/ALS algorithm in the traditional chromatographic method was appropriately able to resolve highly drifted background constituents as well as heavily overlapped peaks among the analytes and also between the analytes and the matrix interferences. Recoveries were ranged from 69.6% to 120.3% with relative standard deviations of less than or equal to 11.0% and showed the acceptable performance of the method. Additionally, statistical t-test as well as computed elliptical joint confidence region (EJCR) confirmed the accuracy of the proposed method and indicated the absence of both constant and proportional errors in the predicted concentrations. The results well explained that the second-order advantage for analytes was achieved in samples containing one or more uncalibrated components, which strongly related to wastewater samples.

ELP-z and ELP-zz capturing scaffolds for the purification of immunoglobulins by affinity precipitation

The increasing demand of monoclonal antibodies for therapeutic applications along with the high manufacturing cost have made it necessary to evaluate better process options and technologies for their purification. Affinity precipitation is an attractive alternative to traditional chromatographic methods by affording effective purification using a simple environmental trigger. The feature of elastin-like-protein (ELP) fused with antibody binding domains has already been explored for the purification of antibodies. However, ELP when fused with the bulkier domains such as Protein A, resulted in lower protein production. In this study, ELP was fused to smaller synthetic IgG binding domains such as the z or zz domain, resulting in up to 10-fold higher level of production. Both ELP-z and ELP-zz bind tightly to human immunoglobulin (HIgG) with a dissociation constant of 768±142nM and 68±23nM, respectively. Owing to the higher binding affinity, the use of ELP-zz resulted in more than 99% recovery of HIgG in four repeated binding and elution cycles with no observable decrease in the purification performance. The same binding and elution cycle was successfully implemented for the purification of monoclonal antibodies from hybridoma culture supernatant with close to 100% recovery.

Production of alkaliphilic, halotolerent, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agro waste: single step purification and characterization

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular alkaliphilic, thermostable and halotolerent xylanase. The culture conditions for xylanase production were optimized with respect to pH, temperature, NaCl and inexpensive agro waste as substrates. Xylanase yield was enhanced more than four fold in the presence of 1% corn husk and 0.5% peptone or feather hydrolysate at pH 11 and 37°C. Xylanase was purified to 11.8-fold with 8.7% yield by using traditional chromatographic methods whereas the same enzyme purified to 20-fold with 72% yield by using corn husk as ligand. Its molecular mass was estimated to be 24kDa by SDS-PAGE. The xylanase had maximal activity at pH 11 and 70°C. The enzyme was active over broad range, 0-20% sodium chloride. The enzyme was thermostable retaining 100% of the original activity at 70°C for 3h. The apparent K (m) values for oat spelt xylan and brichwood xylan were 4.1 and 4.4mg/ml respectively. The deduced internal amino acid sequence of PPKS-2 xylanase resembled the sequence of β-1,4-endoxylanase, which is member of glycoside hydrolase family 11.

1H NMR-based metabolic profiling and target analysis: a combined approach for the quality control of Thymus vulgaris

The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d 6 enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents. The acquisition of 600 MHz 1H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches. Preliminary analysis of the 1H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol, carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%, inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation, rapidity and reproducibility of the NMR analysis.