A validated high-performance liquid chromatography method for detecting geniposide, ellagic acid, piperine, costunolide and dehydrocostus lactone in Liuwei Muxiang capsules.

In this study, a sensitive high-performance liquid chromatography detector was established and validated for the simultaneous determination of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone in Liuwei Muxiang Capsules. The analysis was achieved on CHANIN 100-5-C18-H column (5µm, 250 mm×4.6 mm) with the temperature of 30oC. Gradient elution was applied using 0.1% phosphoric acid solution-methanol-acetonitrile (50:50) as mobile phase at the flow rate of 1.0 mL/min. The determination was performed at the wavelength of 225 nm (detecting geniposide), 254 nm (detecting ellagic acid), 343 nm (detecting piperine) and 225 nm (detecting costunolide and dehydrocostuslactone) along with the sample volume of 10µL. The linear ranges of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone demonstrated good linear relationships within their respective determination ranges. The average recoveries were 100.04%, 99.86%, 99.79%, 100.17% and 100.41%, respectively. RSD% was 1.3%, 1.2%, 1.2%, 1.2%, 1.5%, respectively. The developed method was proved to be simple, accurate and sensitive, which can provide a quantitative analysis method for the content determination of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone in Liuwei Muxiang capsules.

Human Milk Endocannabinoid Levels as a Function of Obesity and Diurnal Rhythm.

The endocannabinoid system is involved in the regulation of a variety of physiological and cognitive processes. While the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) have been found in breast milk, their role(s) have yet to be determined. This study determined the normal concentration ranges of endocannabinoids (2-AG and AEA) in breast milk and the influences, if any, of obesity and diurnal rhythms on their levels. Milk samples were collected from 36 breastfeeding mothers at 4-8 weeks postpartum at each feed over a 24-h period, and further stratified into three groups based on body mass index (BMI). The samples were analyzed using liquid chromatography mass spectrometry. AEA was below the limit of detection and 2-AG levels averaged 59.3 ± 18.3 ng/mL (± SD) in women with normal BMI. Wide-ranging 2-AG concentrations in the overweight (65.5 ± 41.9 ng/mL) /obese (66.1 ± 40.6 ng/mL) groups suggest BMI may be a contributing factor influencing its levels. Following a diurnal pattern, there was a significantly higher 2-AG concentration observed during the day, as compared to night time samples. In conclusion, our study clearly suggests that appropriate milk collection and storage conditions are critical. Further, body weight and diurnal rhythm appear to influence levels of 2-AG. Based on these results, future studies are underway to determine what specific roles endocannabinoids may play in human milk and how elevated levels of 2-AG may modulate infant appetite and health.

Development and validation of a rapid high-performance thin-layer chromatographic method for quantification of gallic acid, cinnamic acid, piperine, eugenol, and glycyrrhizin in Divya-Swasari-Vati, an ayurvedic medicine for respiratory ailments.

Divya-Swasari-Vati is a calcium containing polyherbal ayurvedic medicine prescribed for the lung-related ailments observed in the current pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 infections. The formulation is a unique quintessential blend of nine herbs cited in Ayurvedic texts for chronic cough and lung infection. Analytical standardization of herbal medicines is the pressing need of the hour to ascertain the quality compliance. This persuaded us to develop a simple, rapid, and selective high-performance thin-layer chromatographic method for Divya-Swasari-Vati quality standardization. The developed method was validated for the quantification of marker components, gallic acid, cinnamic acid, piperine, eugenol and glycyrrhizin, against reference standards in five different batches of Divya-Swasari-Vati. The analytes were identified by visualization at 254 nm, and by matching their retention factor with authentic standards. The developed method was validated as per the guidelines recommended by the International Council for Harmonization for parameters like, linearity, limit of detection, limit of quantification, accuracy, and precision. Therefore, the developed novel high-performance thin-layer chromatographic process could be employed for rapid standardization of Divya-Swasari-Vati and other related herbal formulation, which would aid in quality manufacturing and product development.

BIONOTE as an Innovative Biosensor for Measuring Endocannabinoid Levels.

In this study, a novel approach was developed to quantify endocannabinoids (eCBs), and was based on the liquid biosensor BIONOTE. This device is composed of a probe that can be immersed in a solution, and an electronic interface that can record a current related to the oxy-reductive reactions occurring in the sample. The two most representative members of eCBs have been analysed in vitro by BIONOTE: anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG). Bovine serum albumin was used to functionalize the probe and improve the sensibility of the whole analytical system. We show that BIONOTE is able to detect both AEA and 2-AG at concentrations in the low nanomolar range, and to discriminate between these eCBs and their moieties arachidonic acid, ethanolamine and glycerol. Notably, BIONOTE distinguished these five different molecules, and it was also able to quantify AEA in human plasma. Although this is just a proof-of-concept study, we suggest BIONOTE as a cheap and user-friendly prototype sensor for high throughput quantitation of eCB content in biological matrices, with an apparent diagnostic potential for tomorrow's medicine.

In-tube solid-phase microextraction directly coupled to tandem mass spectrometry for anandamide and 2-arachidonoylglycerol determination in rat brain samples from an animal model of Parkinson's disease.

To evaluate the endocannabinoid system in an animal model of Parkinson's disease, in-tube solid-phase microextraction (in-tube SPME) was directly coupled to a tandem mass spectrometry (MS/MS) system for determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples. In-tube SPME-which consisted of a microtube of restricted access material (RAM) with a hydrophilic diol external surface and a hydrophobic octyl inner surface-efficiently excluded (up to 95%) macromolecules from the biological samples and selectively pre-concentrated the analytes. In-tube SPME parameters, such as sample volume, mobile phases, flow rate, and pre-concentration time, were evaluated to improve the extraction efficiency and throughput performance. The selectivity of the in-tube SPME and MS/MS (MRM mode) techniques allowed them to be directly coupled online, which dismissed the need for the chromatographic separation step. The in-tube SPME-MS/MS method was validated and shown to be linear from 6.0 to 30.0 ng mL-1 for AEA and from 10.0 to 100.0 ng mL-1 for 2-AG; the intra- and inter-assay accuracy and precision were lower than 15%. Parallelism between the calibration curves constructed in the matrix and aqueous solution confirmed that there was no matrix effect. The method allowed endogenous concentrations of AEA and 2-AG to be determined in rat brain striatum from unilaterally 6-hydroxydopamine-lesioned animals. The concentrations of these endocannabinoids in striatum ipsilateral and contralateral to the lesion differed significantly (p<0.001).

Identification of the metabolites of piperine via hepatocyte incubation and liquid chromatography combined with diode-array detection and high-resolution mass spectrometry.

RATIONALE: Piperine, an alkaloid isolated from Piper nigrum L., has been demonstrated to have many pharmacological effects and several health benefits. The aim of this work was to study the metabolic profiles of piperine in mouse, rat, dog and human hepatocytes. METHODS: The biotransformation was carried out by incubating piperine with hepatocytes at 37°C. After incubation for 2 h, the samples were pretreated and analyzed using liquid chromatography combined with diode-array detection and high-resolution mass spectrometry (LC/DAD-HRMS). The structures of the metabolites were assigned through a comparison of their accurate masses and product ions with those of the parent compound. RESULTS: A total of 20 metabolites were detected, and the structures were proposed. Piperine was metabolized through the following pathways: (a) oxidation to form a catechol derivative, which further underwent methylation, glucuronidation, glutathione (GSH) conjugation, and hydroxylation followed by opening of the piperidine ring; (b) hydroxylation to form a carbinolamine intermediate followed by opening of the piperidine ring and the formation of alcohol and acid derivatives; and (c) hydroxylation to form stable hydroxylated metabolites. In mouse, the formation of the catechol derivative (M12) and hydroxylation (M11) were the major metabolic pathways; in rat, the formation of the catechol derivative (M12) and glucuronidation (M9) were the main pathways; and in dog and human, the formation of the catechol derivative (M12) was the predominant pathway. No human-specific metabolite was observed. CONCLUSIONS: This study provided some new information on the metabolic profiles of piperine, which should be of great importance in the study of the pharmacology and toxicity of this compound.

A micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry to determine anandamide and 2-arachidonoylglycerol in rat brain samples.

A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL-1 for AEA and from 0.300 to 10.00 µg mL-1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD).

Circulating endocannabinoid concentrations in grieving adults.

Bereavement is one of the most intense, distressing, and traumatic events an elderly person will experience. The symptom responses to bereavement vary, particularly during the first year. However, the neurobiology underlying the symptom variance in grief is poorly understood. The endocannabinoid signaling (ECS) system is stress-responsive; mounting evidence implicates the central ECS in psychopathology. The current study aimed to investigate the hypothesis that the ECS is abnormal in grief, using circulating eCB concentrations as a biomarker of central ECS. A predominantly older sample of grief participants, within 13 months following the death of a loved one, and healthy comparison (HC) participants were studied. Associations of circulating eCBs with symptom variance in grievers were also examined. A total of 61 (grief: n = 44; HC: n = 17) adults completed cross-sectional clinical assessments and a fasting blood draw. Assessments included the Inventory of Complicated Grief scale; the 17-item Hamilton Depression Rating Scale; and the Hamilton Anxiety scale. Serum eCB concentrations (i.e., N-arachidonoylethanolamine [AEA] and 2-arachidonoylglycerol [2-AG]) were quantified using isotope dilution, liquid chromatography-mass spectrometry. Relative to HC participants, grievers had significantly elevated serum AEA but similar 2-AG concentrations. In grievers, serum AEA concentrations were positively associated with depressive and anxiety symptoms, but only in those with low grief symptoms. These novel findings indicate that elevated circulating eCB concentrations are found following bereavement. The eCB signaling response varies based on the degree of grief severity. Circulating eCB measures may have the potential to serve as biomarkers of prolonged grief disorder.

Analytical method development and validation of reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous quantifications of quercetin and piperine in dual-drug loaded nanostructured lipid carriers.

Quercetin and piperine are often used as an add-on therapy for various diseases, however both drug exhibits poor aqueous solubility and photosensitivity issue. Therefore, the aim of the present study is to improve the pharmaceutical challenges by incorporating both the drugs in nanostructured lipid carriers (NLCs) and to develop a sensitive, selective, accurate and precise reverse-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous analysis of both drugs in NLCs. Effective chromatographic separation of quercetin and piperine was achieved on Hypersil gold C-18 column and mobile phase consisting of a mixture of acetonitrile and HPLC grade water (pH 2.6, adjusted with 2%v/v glacial acetic acid) in an isocratic elution mode. The flow rate of the mobile phase was 1 mL/min, column temperature at 35 ± 0.2 °C and the injection volume was 20 µL. The retention time for quercetin and piperine were found to be at 2.80 min and 10.36 min, respectively and detected at an isobestic wavelength of 346 nm using a photodiode array (PDA) detector. The method was found to be specific for the simultaneous analysis of quercetin and piperine in presence of NLCs matrix, accurate (>90%) and precise (%RSD < 2%). The validated RP-HPLC method effectively utilised to determine the percentage drug entrapment efficiency cum percentage drug loading of quercetin and piperine in NLCs enriched formulations along with the secondary estimation of in vitro cumulative percentage drug release study. The results were found to be reliable, hence the validated RP-HPLC method could be further used for the simultaneous detection and quantification of both these drugs in other lipid-based nano-formulations such as solid-lipid nanoparticles, polymer-lipid hybrid nanoparticles, lipid drug conjugates, etc. in in vitro and in vivo.

Switchable-hydrophilicity solvent liquid-liquid microextraction versus dispersive liquid-liquid microextraction prior to HPLC-UV for the determination and isolation of piperine from Piper nigrum L.

Switchable-hydrophilicity solvent liquid-liquid microextraction and dispersive liquid-liquid microextraction were compared for the extraction of piperine from Piper nigrum L. prior to its analysis by using high-performance liquid chromatography with UV detection. Under optimum conditions, limits of detection and quantitation were found as 0.2-0.6 and 0.7-2.0 µg/mg with the two methods, respectively. Calibration graphs showed good linearity with coefficients of determination (R2 ) higher than 0.9962 and percentage relative standard deviations lower than 6.8%. Both methods were efficiently used for the extraction of piperine from black and white pepper samples from different origins and percentage relative recoveries ranged between 90.0 and 106.0%. The results showed that switchable-hydrophilicity solvent liquid-liquid microextraction is a better alternative to dispersive liquid-liquid microextraction for the routine analysis of piperine in food samples. A novel scaled-up dispersive liquid-liquid microextraction method was also proposed for the isolation of piperine providing a yield of 102.9 ± 4.9% and purity higher than 98.0% as revealed by NMR spectroscopy.

Selective in vivo molecular and cellular biocompatibility of black peppercorns by piperine-protein intrinsic atomic interaction with elicited oxidative stress and apoptosis in zebrafish eleuthero embryos.

Day to day consumption of black pepper raise concern about the detailed information about their medicinal, pharmaceutical values and knowledge about the biocompatibility with respect to ecosystem. This study investigates the in vivo selective molecular biocompatibility of its seed cover (SC) and seed core (SP) powder extract using embryonic zebrafish model. Gas chromatography mass spectrometry (GCMS) analysis of the extract prepared by grinding showed presence of different components with "piperine" as principle component. Biocompatibility analysis showed dose and time dependent selective effect of SC and SP with LC50 of 30.4 µg/ml and 35.6 µg/ml, respectively on survivability, hatching and heartbeat rate in embryonic zebrafish. Mechanistic investigation elucidated it as effect of accumulation and internalization of black pepper leading to their influence on structure and function of cellular proteins hatching enzyme (he1a), superoxide dismutase (sod1) and tumor protein (tp53) responsible for delayed hatching, oxidative stress induction and apoptosis. The study provided insight to selective biocompatibility of black pepper expedient to produce higher quality spices with respect to pharmaceutical, clinical and environmental aspects.

Essential fatty acids deficient diet modulates N-Acylethanolamide profile in rat's tissues.

No data are available on whether a diet deficient of the essential fatty acids is able to modulate tissue levels of endocannabinoids and congeners. Male rats fed for 12 weeks a diet deficient of essential fatty acids, palmitic and oleic acids (EFAD), replaced with saturated fatty acids (SAFA), showed lowered n-3 and n-6 PUFAs levels in plasma, liver and adipose tissue, with concomitant steep increase of oleic and mead acids, while in hypothalamus no changes in PUFA concentration were detected and only palmitoleic acid was found increased. We found a reduction of anandamide and palmitoylethanolamide in liver and brain, while oleoylethanolamide increased significantly in liver and adipose tissue, associated to a 50 % body weight decrease. Changes in N-acylethanolamide profile may contribute to body weight reduction distinctive of EFA deficiency.

Characteristics, Biological Properties and Analytical Methods of Piperine: A Review.

Piperine (PIP) is a natural alkaloid isolated from Piper longum L. that presents antioxidant, anticonvulsant, antimicrobial, neuroprotective, larvicidal, antiparasitic, anticancer effect and other pharmacological properties. However, the low aqueous solubility is the main barrier to its development from the laboratory to the clinic as a drug. Several strategies have been used to overcome this obstacle, like the incorporation of PIP into different drug delivery systems turned out to be highly efficient. In addition, several methods for the quantitative and qualitative analysis of PIP in various raw materials, including biological fluids (plasma, urine, metabolites, brain), plants and drug delivery systems, were investigated. Most recently high-performance liquid chromatography was used together with several detectors for this purpose. Therefore, this review presents a summary of characteristics chemical and biological properties of PIP as well as several techniques and analytical methods to optimize the analytical signal, increase sensitivity, selectivity and reduce the effects of interference for this drug.

Antifungal and anti-aflatoxigenic activity of Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus by downregulating the expression of alfD and aflR genes of the aflatoxins biosynthetic pathway.

In the present study, ethanolic extract from Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus growth and aflatoxins production were studied in relation to the expression of aflD and aflR, two key genes of aflatoxins biosynthetic pathway. Phytochemical analysis of the ethanolic extract by GC-EIMS identified affinin/spilanthol (7.84 ± 0.27 mg g-1) as the most abundant compounds in H. longipes roots. The antifungal and anti-aflatoxigenic assays showed that affinin/spilanthol at 300 µg mL-1 produced the higher inhibition of radial growth (95%), as well as, the higher aflatoxins production inhibition (61%) in comparison to H. longipes roots (87% and 48%, respectively). qRT-PCR revealed that the expression of aflD and aflR genes showed a higher downregulation in affinin/spilanthol at 300 µg mL-1. The expression ratio of alfD was suppressed by affinin/spilanthol in 79% and aflR in 84%, while, a lower expression ratio suppressed by H. longipes was obtained, alfD (55%) and aflR (59%). Affinin/spilanthol possesses higher antifungal and anti-aflatoxigenic activity against A. parasiticus rather than H. longipes roots, and this anti-aflaxotigenic activity occurring via downregulation of the aflD and aflR genes. Thus, H. longipes roots and affinin/spilanthol can be considered potent antifungal agents against aflatoxigenic fungus, especially, affinin/spilanthol.

Electrochemical quantification of piperine in black pepper.

A simple, rapid method of the detection of piperine in black pepper is reported using a voltammetric sensor based on a glassy carbon (GC) electrode with analysis following a short one-step extraction using ethanol. The method is based on a novel potential sweep designed to maximise signal sizes and shown with context of the present analytical challenge to be essential for gathering data allowing the construction of a linear calibration curve for the analysis in the relevant range namely 0.25-5.0 mM.

Simultaneous determination of six components in commercial Roukou Wuwei pills using ultra-high-performance liquid chromatography with diode-array detector.

An efficient ultra-performance liquid chromatography with diode-array detector method was established for simultaneous determination of six active components in Roukou Wuwei pills, namely gallic acid, piperine, costundide, dehydrocostus lactone, isoalantolactone and alantolactone. Chromatographic separation of six components was successfully achieved on an Waters BEH C18 column (50 × 2.1 mm, 1.7 µm) with a mobile phase composed of acetonitrile and water using a gradient elution. Gallic acid and piperine were detected at 270 nm and 343 nm, respectively; while costundide, dehydrocostus lactone, isoalantolactone and alantolactone were simultaneously measured at 225 nm. All six calibration curves showed good linearity (R2 ≥ 0.9994) between the peak area of each component and corresponding concentration. Relative standard deviations for inter- and intra-day precisions were <0.45 and 0.77%, respectively. The mean recovery rates ranged from 96.72 to 102.2% with relative standard deviations <2.07%. The developed method was validated in terms of linearity, precision and accuracy and then successfully applied for the quality control of commercial Roukou Wuwei samples.

Characterization of stable and reactive metabolites of piperine formed on incubation with human liver microsomes.

Black pepper, though commonly employed as a spice, has many medicinal properties. It consists of volatile oils, alkaloids, pungent resins, etc., of which piperine is a major constituent. Though safe at low doses, piperine causes alteration in the activity of drug metabolising enzymes and transporters at high dose and is known to precipitate liver toxicity. It has a potential to form reactive metabolite(s) (RM) owing to the presence of structural alerts, such as methylenedioxyphenyl (MDP), α, ß-unsaturated carbonyl group (Michael acceptor), and piperidine. The present study was designed to detect and characterize stable and RM(s) of piperine formed on in vitro incubation with human liver microsomes. The investigation of RMs was done with the aid of trapping agents, viz, glutathione (GSH) and N-acetylcysteine (NAC). The samples were analysed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) using Thermo Scientific Q Exactive Plus Orbitrap. Full scan MS followed by data-dependent MS2 (Full MS-ddMS2 ) mode was used to establish mass spectrometric fragmentation pathways of protonated piperine and its metabolites. In total, four stable metabolites and their isomers (M1a-c, M2a-b, M3a-c, and M4a-b) were detected. Their formation involved removal of carbon (3, M1a-c), hydroxylation (2, M2a-b), hydroxylation with hydrogenation (3, M3a-c), and dehydrogenation (2, M4a-b). Out of these metabolites, M1, M2, and M3 are reported earlier in the literature, but their isomers and two M4 variants are novel. In addition, six novel conjugates of RMs, including three GSH conjugates of m/z 579 and three NAC conjugates of m/z 435, were also observed.

Phytochemical and pharmacological attributes of piperine: A bioactive ingredient of black pepper.

Plants are vital for the wellbeing of humankind in a variety of ways. Some plant extracts contain antimicrobial properties that can treat different pathogens. Most of the world's population relies on medicinal plants and natural products for their primary health care needs. Therefore, there is a growing interest in natural products, medicinal plants, and traditional medicine along with a desire to design and develop novel plant-based pharmaceuticals. These plant-based pharmaceuticals may address the concerns of reduced efficacy of synthetic antibiotics due to the emergence of drug-resistant pathogens. In this regard, some plant extracts from black pepper (Piper nigrum) with antimicrobial properties, including piperine, have the potential to be used as natural dietary supplements together with modern therapeutic approaches. This review highlights possible applications of piperine as the active compound in the fields of rational drug design and discovery, pharmaceutical chemistry, and biomedicine. We discuss different extraction methods and pharmacological effects of the analyzed substance to pave the way for further research strategies and perspectives towards the development of novel herbal products for better healthcare solutions.

Members of the endocannabinoid system are distinctly regulated in inflammatory bowel disease and colorectal cancer.

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.

[Quantitative Analysis of Piperine and the Derivatives in Long Pepper Extract by HPLC Using Relative Molar Sensitivity].

A novel method was developed for quantification of five major piperine derivatives (piperanine, piperine, chavicine, isopiperine, and isochavicine) in a hot water extract of long pepper fruit (LPE) using the relative molar sensitivity (RMS) based on the combination of HPLC/UV and 1H- quantitative NMR (1H-qNMR). The RMSs of piperanine, chavicine, isopiperine, and isochavicine to piperine of which the absolute purity was determined by 1H-qNMR were calculated to be 0.3693, 1.138, 0.9164, and 1.277, respectively. The total amount of piperine derivatives in LPE was quantified by both 1H-qNMR and HPLC/UV based on the RMS using piperine as a single-reference material (RMS method). The relative difference in quantitation values of 1H-qNMR and calibration curve method from the RMS method was 2.01% or less. The relative difference of the total cis-trans piperine isomers content between before and after photoirradiation in piperine solution was quantified to be 2.84% by the RMS method. In addition, the interlaboratory difference of the RMS method was confirmed in the range of 0.600 to 4.00 µg/g when analysis was performed on piperine derivatives in LPE containing tablets, while the total amount of piperine derivatives in the tablets was quantified at 606 µg/g. Our proposed method is a reliable tool for determining the contents of piperine and the derivatives in LPE and processed foods containing LPE.