Effect of woohwangchungsimwon and donepezil co-treatment on cognitive function and serum metabolic profiles in a scopolamine-induced model of Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Woohwangchungsimwon (WCW) is a traditional medicine used in East Asian countries to treat central nervous system disorders. Reported pharmacological properties include antioxidant effects, enhanced learning and memory, and protection against ischemic neuronal cell death, supporting its use in treating neurodegenerative diseases like Alzheimer's disease (AD). AIM OF THE STUDY: The study aims to assess the effects of co-treatment with WCW and donepezil on cognitive functions and serum metabolic profiles in a scopolamine-induced AD model. MATERIALS AND METHODS: Cell viability and reactive oxygen species (ROS) levels were measured in amyloid ß-peptide25-35 (Aß25-35)-induced SH-SY5Y cells. An AD model was established in ICR mice by intraperitoneal scopolamine administration. Animals underwent the step-through passive avoidance test (PAT) and Morris water maze (MWM) test. Hippocampal tissues were collected to examine specific protein expression. Serum metabolic profiles were analyzed using nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Co-treatment with WCW and donepezil increased cell viability and reduced ROS production in Aß25-35-induced SH-SY5Y cells compared to that with donepezil treatment alone. Co-treatment improved cognitive functions and was comparable to donepezil treatment alone in the PAT and MWM tests. Pathways related to tyrosine, phenylalanine, and tryptophan biosynthesis, phenylalanine metabolism, and cysteine and methionine metabolism were altered by co-treatment. Levels of tyrosine and methionine, major serum metabolites in these pathways, were significantly reduced after co-treatment. CONCLUSIONS: Co-treatment with WCW and donepezil shows promise as a therapeutic strategy for AD and is comparable to donepezil alone in improving cognitive function. Reduced tyrosine and methionine levels after co-treatment may enhance cognitive function by mitigating hypertyrosinemia and hyperhomocysteinemia, known risk factors for AD. The serum metabolic profiles obtained in this study can serve as a foundation for developing other bioactive compounds using a scopolamine-induced mouse model.

Efficacy and Safety of Woohwangchungsimwon Combined with Donepezil in Behavioral and Psychological Symptoms of Dementia in Patients with Probable Alzheimer's Disease: Study Protocol for a Randomized Controlled Trial.

Behavioral and psychological symptoms of dementia are a major factor in the burden of care and medical expenses. Conventional pharmacological treatments do not exert a distinct effect on the benefits versus the risks. The herbal medicine woohwangchungsimwon is frequently prescribed for neuropsychiatric disorders. An effect of woohwangchungsimwon on behavioral and psychological symptoms of dementia has been previously reported; however, no clinical studies have been conducted. We aim to evaluate the efficacy and safety of woohwangchungsimwon combined with donepezil for alleviating these symptoms in probable Alzheimer's disease. In this randomized, assessor-blinded, parallel-group clinical trial, 74 participants with probable Alzheimer's disease will be divided via block randomization into a woohwangchungsimwon + donepezil combination group (n = 37) or a donepezil single group (n = 37). Participants will include patients under donepezil treatment for at least a month. We will perform the study for 24 weeks. The Neuro-Psychiatric Inventory subscale scores will be the primary outcome. Secondary outcomes will include cognitive function, dementia severity, physical function, quality of life, depression, anxiety, and insomnia. For safety evaluation, we will assess adverse reactions, measure vital signs, and conduct laboratory tests. This is the first trial aiming to confirm the efficacy and safety of woohwangchungsimwon combined with donepezil for alleviating behavioral and psychological symptoms of dementia. Its findings could provide a basis for their co-administration to control these symptoms in probable Alzheimer's disease.

1H-NMR-Based Metabolic Profiling of Cordyceps militaris to Correlate the Development Process and Anti-Cancer Effect.

The study of metabolomics in natural products using the diverse analytical instruments including GC-MS, LC-MS, and NMR is useful for the exploration of physiological and biological effects and the investigation of drug discovery and health functional foods. Cordyceps militaris has been very attractive to natural medicine as a traditional Chinese medicine, due to its various bioactive properties including anti-cancer and anti-oxidant effects. In this study, we analyzed the metabolite profile in 50% ethanol extracts of C. militaris fruit bodies from three development periods (growth period, matured period, and aging period) using 1H-NMR, and identified 44 metabolites, which are classified as 16 amino acids, 10 organic acids, 5 carbohydrates, 3 nucleotide derivatives, and 10 other compounds. Among the three development periods of the C. militaris fruit body, the aging period showed significantly higher levels of metabolites including cordycepin, mannitol (cordycepic acid), and ß-glucan. Interestingly, these bioactive metabolites are positively correlated with antitumor growth effect; the extract of the aging period showed significant inhibition of HepG2 hepatic cancer cell proliferation. These results showed that the aging period during the development of C. militaris fruit bodies was more highly enriched with bioactive metabolites that are associated with cancer cell growth inhibition.

Changes in fluorescent dissolved organic matter and their association with phytoavailable phosphorus in soil amended with TiO2 nanoparticles.

This study presents the impacts of TiO2 nanoparticles (TNPs) amendment on plant growth, phosphorus (P) content, and dissolved organic matter (DOM) composition in the rhizosphere. For this work, wheat plants (Galaxy-2013) were exposed to soil amended by different amounts of TNPs (i.e., 0, 50, and 100 mg TNP/kg of soil) for 40 days and harvested. The maximum increase in the shoots and roots lengths reached 15.9 ±â€¯0.3% and 3.8 ±â€¯0.3% respectively, which was concurrent with improved P content in the plants. Compared with the control, the P content in the shoots and roots was enriched by 23.4% and 17.9% at 50 mg TNP/kg of soil respectively. The increased electrical conductivity (EC) and decreased pH of the rhizosphere implied that the added TNPs might induce the enhancement of the P dissolution. Fluorescence spectroscopy revealed the increase of microbial activity as depicted by the humification index (HIX) changing from 0.88 ±â€¯0.02 to 0.92 ±â€¯0.01, with increasing TNPs amendments. Excitation-emission matrix coupled with parallel factor analysis (EEM-PARAFAC) showed the presence of four fluorescent components (C1 to C4) in the rhizosphere. Three of them (C1-C3) were related to humic-like substances, while the C4 was associated with protein-like fluorescence. EEM-PARAFAC results revealed the degradation of C4, and the enhancement of the other three components, which supported the stimulation of microbial activity by the TNPs amendment. This study provided new insights into the relation between improved phytoavailble P in plants and the changes in the rhizosphere soil solution chemistry and the DOM composition upon TNPs amendments.

Chemical profiles and antioxidant properties of roasted rice hull extracts in bulk oil and oil-in-water emulsion.

We conducted in vitro assays to investigate the antioxidant properties of aqueous or ethanolic extracts of roasted rice hulls. Phenolic compound contents were analyzed via HPLC, and comprehensive chemical profiles were obtained via 1H NMR and 1H-H COSY NMR. Rice hulls were roasted at 150, 180, and 230 °C for 20 min, after which the aqueous and 70% ethanol extracts were obtained. In vitro assays showed that rice hulls roasted at 230 °C possessed very strong antioxidant properties (p < 0.05). Comprehensive chemical profiles determined via NMR showed that roasting increased the contents of specific amino acids and monosaccharides and reduced the organic acid contents. p-Coumaric, vanillic, and ferulic acids were the most dominant phenolic compounds present in the sample. Added roasted rice hull extracts enhanced the oxidative stability of bulk oil and in O/W emulsions at 60 °C. However, the extracts accelerated lipid oxidation rates of oils heated at 180 °C.

Metabolomics and Lipidomics Approaches in the Science of Probiotics: A Review.

The intestinal microflora plays important roles in the health of the host, such as nutrient processing and the modulation of intestinal immune responses. The constituents of the diet greatly affect the composition of the microbiota and its metabolites. The human intestinal microbiota is made up of around 100 trillion microbial cells encompassing at least 300 species. Consuming probiotics may lead to changes in the intestinal microflora that influence host health. Metabolomics is a powerful tool for revealing metabolic changes in biofluids, tissues, and organs of hosts induced by the consumption of probiotics, and lipidomics in particular is a technical approach that focuses on the analysis of lipids in various cells and biofluids. Metabolomics and lipidomics have been used to investigate intracellular and extracellular metabolites as well as for the nontargeted profiling and fingerprinting of metabolites. Based on metabolomics and lipidomics investigations, we reviewed the effects of consuming probiotics on metabolic profiles in controlled intestinal environments. We also discuss the associations between metabolic changes and human diseases after consuming probiotics in uncontrolled intestinal environments. In addition, we review the metabolic changes that take place within the food matrix during probiotic fermentation.

Discrimination and prediction of cultivation age and parts of Panax ginseng by Fourier-transform infrared spectroscopy combined with multivariate statistical analysis.

Panax ginseng C.A. Meyer is a herb used for medicinal purposes, and its discrimination according to cultivation age has been an important and practical issue. This study employed Fourier-transform infrared (FT-IR) spectroscopy with multivariate statistical analysis to obtain a prediction model for discriminating cultivation ages (5 and 6 years) and three different parts (rhizome, tap root, and lateral root) of P. ginseng. The optimal partial-least-squares regression (PLSR) models for discriminating ginseng samples were determined by selecting normalization methods, number of partial-least-squares (PLS) components, and variable influence on projection (VIP) cutoff values. The best prediction model for discriminating 5- and 6-year-old ginseng was developed using tap root, vector normalization applied after the second differentiation, one PLS component, and a VIP cutoff of 1.0 (based on the lowest root-mean-square error of prediction value). In addition, for discriminating among the three parts of P. ginseng, optimized PLSR models were established using data sets obtained from vector normalization, two PLS components, and VIP cutoff values of 1.5 (for 5-year-old ginseng) and 1.3 (for 6-year-old ginseng). To our knowledge, this is the first study to provide a novel strategy for rapidly discriminating the cultivation ages and parts of P. ginseng using FT-IR by selected normalization methods, number of PLS components, and VIP cutoff values.

State of Panax ginseng Research: A Global Analysis.

This article aims to understand the global and longitudinal trends of research on Panax ginseng. We used bibliometrics to analyze 3974 papers collected from the Web of ScienceTM Core Collection database during 1959-2016. The number of publications showed a steady growth before 2000 and exponentially increased in stage III (2000-2016, about 86% of the papers were published). Research on P. ginseng was conducted in 64 countries, mainly in Asia; in particular, 41% and 28% of the publications were from South Korea and China, respectively. The institutions from South Korea and China had high publication output and close cooperation and provided the majority of financial support. All top 10 authors and four of the top 20 journals in terms of number of publications originated from South Korea. The leading research subjects were pharmacology (39%), plant science (26%), and integrative complementary medicine (19%). The hotspot of P. ginseng research transformed from basic science to application, and multidisciplinary sciences will play a substantial role in the future. This study provides a comprehensive analysis to elucidate the global distribution, collaboration patterns, and research trends in the P. ginseng domain.

Growth and Metabolic Responses of Rice (Oryza sativa L.) Cultivated in Phosphorus-Deficient Soil Amended with TiO2 Nanoparticles.

Plants have the natural ability to withstand stress conditions through metabolic adjustments. The present study aimed at investigating the effects of titanium dioxide nanoparticles (TiO2 NPs) application (0, 25, 50, 150, 250, 500, and 750 mg kg-1) in phosphorus-deficient soil in terms of growth responses, P contents, and metabolic alterations in rice. TiO2 NPs application increased shoot length up to 14.5%. Phosphorus contents in rice roots, shoots, and grains were increased by 2.6-, 2.4-, and 1.3-fold, respectively, at 750 mg kg-1 of TiO2 NPs. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics revealed increased levels of amino acids, palmitic acid, and glycerol content in grains resulting from plants grown in 750 mg kg-1 TiO2 NPs-treated soil. Furthermore, no translocation of TiO2 NPs from the treated soil to rice grains was detected by inductively coupled plasma-optical emission spectrometry (ICP-OES), which suggests no risk of TiO2 NPs intake via grain consumption. The observed data indicates the strong relationship among NPs application, P contents, and metabolic alterations.

NanoESI-MS-based lipidomics to discriminate between cultivars, cultivation ages, and parts of Panax ginseng.

Korean ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs used in Asia, including Korea and China. In the present study lipid profiling of two officially registered cultivars (P. ginseng 'Chunpoong' and P. ginseng 'Yunpoong') was performed at different cultivation ages (5 and 6 years) and on different parts (tap roots, lateral roots, and rhizomes) using nano-electrospray ionization-mass spectrometry (nanoESI-MS). In total, 30 compounds including galactolipids, phospholipids, triacylglycerols, and ginsenosides were identified. Among them, triacylglycerol 54:6 (18:2/18:2/18:2), phosphatidylglycerol 34:3 (16:0/18:3), monogalactosyldiacylglycerol 36:4 (18:2/18:2), phosphatidic acid species 36:4 (18:2/18:2), and 34:1 (16:0/18:1) were selected as biomarkers to discriminate cultivars, cultivation ages, and parts. In addition, an unknown P. ginseng sample was successfully predicted by applying validated partial least squares projection to latent structures regression models. This is the first study regarding the identification of intact lipid species from P. ginseng and to predict cultivars, cultivation ages, and parts of P. ginseng using nanoESI-MS-based lipidomic profiling with a multivariate statistical analysis.

Chemical analysis of Panax quinquefolius (North American ginseng): A review.

Panax quinquefolius (PQ) is one of the best-selling natural health products due to its proposed beneficial anti-aging, anti-cancer, anti-stress, anti-fatigue, and anxiolytic effects. In recent years, the quality of PQ has received considerable attention. Sensitive and accurate methods for qualitative and quantitative analyses of chemical constituents are necessary for the comprehensive quality control to ensure the safety and efficacy of PQ. This article reviews recent progress in the chemical analysis of PQ and its preparations. Numerous analytical techniques, including spectroscopy, thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS), high-speed centrifugal partition chromatography (HSCPC), high-performance counter-current chromatography (HPCCC), nuclear magnetic resonance spectroscopy (NMR), and immunoassay, are described. Among these techniques, HPLC coupled with mass spectrometry (MS) is the most promising method for quality control. The challenges encountered in the chemical analysis of PQ are also briefly discussed, and the remaining questions regarding the quality control of PQ that require further investigation are highlighted.

Antiproliferative and Apoptotic Activity of Chamaecyparis obtusa Leaf Extract against the HCT116 Human Colorectal Cancer Cell Line and Investigation of the Bioactive Compound by Gas Chromatography-Mass Spectrometry-Based Metabolomics.

Chamaecyparis obtusa (CO) belongs to the Cupressaceae family, and it is found widely distributed in Japan and Korea. In this study, the anti-proliferative activities of the methanol and water extracts of CO leaves against a human colorectal cancer cell line (HCT116) were investigated. The methanol extract of CO leaves, at a concentration of 1.25 µg/mL, exhibited anti-proliferative activity against HCT116 cells, while displaying no cytotoxicity against Chang liver cells. Comparative global metabolite profiling was performed using gas chromatography-mass spectrometry coupled with multivariate statistical analysis, and it was revealed that anthricin was the major compound contributing to the anti-proliferative activity. The activation of c-Jun N-terminal kinases played a key role in the apoptotic effect of the methanol extract of CO leaves in HCT116 human colon cancer cells. These results suggest that the methanol extract and anthricin derived from CO leaves might be useful in the development of medicines with anti-colorectal cancer activity.

Metabolomic Analysis Reveals Cyanidins in Black Raspberry as Candidates for Suppression of Lipopolysaccharide-Induced Inflammation in Murine Macrophages.

The extracts produced by multisolvent extraction and subfractionation with preparative liquid chromatography of black raspberry (Rubus coreanus Miquel) cultivated in Gochang, South Korea, were tested for their anti-inflammatory effects. The metabolomic profiling and analysis by orthogonal partial least-squares discriminant analysis (OLPS-DA) suggested that cyanidin, cyanidin-3-glucoside (C3G), and cyanidin-3-rutinoside (C3R) were key components for the anti-inflammatory responses in the most active fraction BF3-1, where they were present at 0.44, 1.26, and 0.56 µg/mg of BF3-1, respectively. Both BF3-1 and mixture of these cyanidins at the same ratio reduced lipopolysaccharide (LPS)-induced protein level of iNOS expression and suppressed mRNA and protein expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß through inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs) and STAT3 in murine macrophage RAW264.7 cells. Overall, the results suggested that co-administration of cyanidin, C3G, and C3R is more effective than that of cyanidin alone and that the coexistence of these anthocyanin components in black raspberry plays a vital role in regulating LPS-induced inflammation even at submicromolar concentrations, making it possible to explain the health beneficial activity of its extracts.

Classification of ginseng berry (Panax ginseng C.A. MEYER) extract using 1H NMR spectroscopy and its inhibition of lipid accumulation in 3 T3-L1 cells.

BACKGROUND: Panax ginseng is a famous traditional medicine in Korea for its beneficial effect on obesity, cardiac and liver associated diseases. The aim of this study was to investigate the metabolite in Panax ginseng (P. ginseng, Aralicaceae) berries depending on the ripen stages and evaluate its potential inhibition on adipocyte differentiation in 3 T3-L1 cells. METHODS: Different ripening stage samples of P. ginseng berry were analyzed through global metabolite profiling by NMR spectroscopy. Lipid accumulation in the cells was analyzed by Oil Red O staining. RESULTS: The PLS-DA clearly distinguished P. ginseng berry extract (PGBE) according to the partial ripe (PR), ripe(R) and fully ripe (FR) stage. Lipid accumulation of PGBE was examined by measuring triglyceride content and Oil-Red O staining. These results suggested that the FR stage of PGBE decrease in lipid accumulation during adipocyte differentiation and the amount of threonine, asparagine, fumarate, tyraine, tyrosine, and phenylalanine increased with longer ripening of ginseng berries. CONCLUSION: Metabolite profiling of P. ginseng was identified by 1H NMR spectra. P. ginseng extract efficiently inhibits adipogenesis in 3 T3-L1 adipocytes concluded that the P. ginseng has the antiobesity properties.

Anti-inflammatory effects of Rubus coreanus Miquel through inhibition of NF-κB and MAP Kinase.

BACKGROUND/OBJECTIVES: Rubus Coreanus Miquel (RCM), used as a traditional Korean medicine, reduces chronic inflammatory diseases such as cancer and rheumatoid arthritis. However, its mechanism has not been elucidated. In this study, we examine the anti-inflammatory effects of RCM and their possible mechanisms using RAW 264.7 cells. MATERIALS/METHODS: Unripe RCM ethanol extract (UE), unripe RCM water extract (UH), ripe RCM ethanol extract (RE), and ripe RCM water extract (RH) were prepared. Inflammatory response was induced with LPS treatment, and expression of pro-inflammatory mediators (iNOS, COX-2, TNF-α, IL-1ß, and IL-6) and NO and PGE2 productions were assessed. To determine the anti-inflammatory mechanism of RCM, we measured NF-κB and MAPK activities. RESULTS: UE and UH treatment significantly reduced NF-κB activation and JNK and p38 phosphorylation and reduced transcriptional activities decreased iNOS, COX-2, and pro-inflammatory cytokines expressions, and NO and PGE2 productions. RE and RH treatments reduced IL-1ß and IL-6 expressions through suppressions of JNK and p38 phosphorylation. CONCLUSIONS: In this study, we showed that RCM had anti-inflammatory effects by suppression of pro-inflammatory mediator expressions. Especially, unripe RCM showed strong anti-inflammatory effects through suppression of NF-κB and MAPK activation. These findings suggest that unripe RCM might be used as a potential functional material to reduce chronic inflammatory responses.

Ellagic acid identified through metabolomic analysis is an active metabolite in strawberry ('Seolhyang') regulating lipopolysaccharide-induced inflammation.

This study employed the metabolomic approach to identify the key constituent exerting anti-inflammatory activity in murine macrophage RAW 264.7 cells. Among the six different fractions (SF1-SF6) of the strawberry 'Seolhyang', SF4 showed more significant inhibition on iNOS expression than SF3, and ellagic acid was determined as the most significant different component between SF4 and SF3 using orthogonal partial least-squares discriminant analysis. Ellagic acid (0.3 and 1.0 µM) and SF4 (100 µg/mL) were found to regulate the same inflammatory mediators, inhibitory κB (IκB) and mitogen-activated protein kinases (MAPKs), which led to the reduction of tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), and iNOS expressions. These results demonstrate that ellagic acid from strawberry 'Seolhyang' is the major component playing a crucial role in inflammation, suggesting the possible application of metabolomic analysis to determining the key ingredients having biological functions in the complicated food matrix.

Alteration of media composition and light conditions change morphology, metabolic profile, and beauvericin biosynthesis in Cordyceps bassiana mycelium.

Metabolic alterations of Cordyceps bassiana mycelium were investigated under the following culture medium and light conditions: dextrose agar supplemented with 0.5% yeast extract (SDAY) medium with light (SL), SDAY medium without light (SD), nut medium without light (ND), and iron-supplemented SDAY medium without light (FD). The levels of asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, ornithine, and proline were significantly higher under SD and SL conditions. The levels of most of the alcohols, saturated fatty acids, unsaturated fatty acids, fatty acid esters, sterols, and terpenes were higher under the ND condition than in the other conditions, but beauvericin was not detectable under the ND condition. The FD condition was favorable for the enhanced production of aminomalonic acid, malic acid, mannonic acid, and erythritol. Thus, the metabolic characteristics of C. bassiana can be manipulated by varying the cultivation conditions, rendering this fungus potentially favorable as a nutraceutical and medicinal resource.

Gas chromatography/mass spectrometry-based metabolic profiling and differentiation of ginseng roots according to cultivation age using variable selection.

Ginseng roots are an important herbal resource worldwide, and the adulteration of ginseng with age is recognized as a serious problem. It is therefore crucial to develop objective criteria or standard protocols for differentiating ginseng root samples according to their cultivation age. The reported study used GC/MS combined with multivariate statistical analysis with variable selection to obtain metabolic profiling and an optimal partial least squares-discriminant analysis (PLS-DA) model for the differentiation of ginseng according to cultivation age. Relative levels of various metabolites, such as amino acids, alcohols, fatty acids, organic acids, and sugars, were measured for various ginseng cultivation ages. Increasing cultivation age resulted in the production of higher levels of panaxynol and panaxydol, which are active polyacetylene compounds in ginseng. In addition, optimized PLS-DA models for the prediction of ginseng age were obtained by selecting variables based on a variable importance in the projection cut-off value of 1.3. Proline, glucaric acid, mannose, gluconic acid, glucuronic acid, myoinositol, panaxydol, and panaxynol are suggested as key and relevant compounds with which to differentiate the age of ginseng samples. The findings of this study suggest that GC/MS-based metabolic profiling can be used to differentiate ginseng samples according to cultivation age.

Nontargeted metabolomics approach for age differentiation and structure interpretation of age-dependent key constituents in hairy roots of Panax ginseng.

The age of the ginseng plant has been considered as an important criterion to determine the quality of this species. For age differentiation and structure interpretation of age-dependent key constituents of Panax ginseng, hairy root (fine root) extracts aged from four to six years were analyzed using a nontargeted approach with ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS). Various classification methods were used to determine an optimal method to best describe ginseng age by selecting influential metabolites of different ages. Through the metabolite selection process, several age-dependent key constituents having the potential to be biomarkers were determined, and their structures were identified according to tandem mass spectrometry and accurate mass spectrometry by comparing them with an in-house ginsenoside library and with literature data. This proposed method applied to the hairy roots of P. ginseng showed an improved efficiency of age differentiation when compared to previous results on the main roots and increases the possibility of the identification of key metabolites that can be used as biomarker candidates for quality assurance in ginseng.

Simple preparative gas chromatographic method for isolation of menthol and menthone from peppermint oil, with quantitative GC-MS and (1) H NMR assay.

The quantitative performance of a simple home-built preparative gas chromatography (prep-GC) arrangement was tested, incorporating a micro-fluidic Deans switch, with collection of the target compound in a deactivated uncoated capillary tube. Repeat injections of a standard solution and peppermint sample were made into the prep-GC instrument. Individual compounds were eluted from the trapping capillary, and made up to constant volume. Chloronaphthalene internal standard was added in some cases. Recovered samples were quantitatively assayed by using GC-MS. Calibration linearity of GC-MS for menthol standard area response against number of injections (2-20 repeat injections) was excellent, giving R(2) of 0.996. For peppermint, menthol correlation over 2-20 repeated injections was 0.998 for menthol area ratio (versus IS) data. Menthone calibration for peppermint gave an R(2) of 0.972. (1) H NMR spectroscopy was conducted on both menthol and menthone. Good correspondence with reference spectra was obtained. About 80 µg of isolated menthol and menthone solute was collected over a sequence of 80 repeat injections from the peppermint sample, as assayed by 600 MHz (1) H NMR analysis (∼100% recovery for menthol from peppermint). A procedure is proposed for prediction of number of injections required to acquire sufficient material for NMR detection.