Adulteration Of Herbal Products Research Articles

Detection of synthetic lipid‐lowering adulterants in herbal products using high‐performance thin‐layer chromatography

AbstractAdulterating herbal medicine with undeclared synthetic drugs is a worldwide problem that seriously threatens human health. To detect such illicit and not labeled synthetic hypolipidemic substances in herbal medicines, an analytical method to simultaneously identify and quantify atorvastatin (ATV) calcium, simvastatin (SIM), gemfibrozil (GEM), fenofibrate (FEN) belonging to two different classes of drugs (statins and fibrates) was developed based on high‐performance thin‐layer chromatography. The method consisted of Silica Gel G60 F254 precoated aluminum plate as stationary phase and toluene: ethyl acetate: formic acid (7:3:0.3, v/v/v) as optimized mobile phase. The densitometric evaluation wavelength was optimized at 254 nm. The Rf values of ATV, SIM, GEM, and FEN were 0.157, 0.268, 0.691, and 0.852, respectively. The linearity range is between 100–1500 ng/band for ATV and FEN and 200–3000 ng/band for SIM and GEM, respectively. The accuracy was found to be in the range of 97%–105%. The relative standard deviation of precision and robustness was found to be < 5%. However, the five screened market samples did not show the presence of adulteration of the above drugs. This method for the simultaneous determination of four drugs may be successfully employed to screen synthetic hypolipidemics as adulterants in herbal products and dietary supplements.

Simultaneous analysis of 4 specific antihypertensive adulterants in herbal products using LC-MS/MS

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous analysis of 4 compounds used for treating hypertension as adulterants in herbal products. The method was validated in terms of selectivity, linearity, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and precision in accordance with AOAC and ICH guidelines. The LOD and LOQ of all compounds were about 0.3 ng/mL and 1 ng/mL, respectively. The linearity was good (R2 > 0.999), with intra-day and inter-day precision levels not more than 9.85% and 8.51%, respectively, and 81.9 – 109.8% accuracies. Thirty commercial herbal products consisting of traditional medicines and dietary supplements available in Vietnamese markets were tested. While none contained detectable amounts of the 4 antihypertensive compounds, the developed LC-MS/MS procedure can be used for routine analysis to monitor illegal adulteration in various forms of herbal products.

Quality control of marketed herbal products of Asparagus racemosus Willd. through high performance thin layer chromatography (HPTLC) analysis

Asparagus racemosus Willd. is a valuable medicinal plant which is used all over the world. There are several marketed products of A. racemosus. The high demand for this herb has increased the risk of adulteration in its commercial products. The adulterated herbal products might pose serious ill effects on health. Therefore, it is necessary to check the quality of marketed products in terms of the presence of their major bioactive compounds. The present study aimed to carry out the qualitative and quantitative analysis of Shatavarin IV in marketed products of A. racemosus through a validated high performance thin layer chromatography (HPTLC) method. Ten marketed products were analysed and all of them had shown the presence of Shatavarin IV which was quantified. The identification and quantification were done by taking a standard Shatavarin IV as reference. The Shatavarin IV was detected at Rf 0.4±0.05 and showed maximum absorption at 425 nm. The Shatavarin IV was quantified using a 6-point calibration curve having a standard deviation of 3.89 % with an R2 value of 0.9968. The amount of Shatavarin IV varied between 1.47±0.25 to 2.69±0.51 mg/g on a dry weight basis which is a normal range in the raw plant materials. Thus, the present findings would be a simple, reliable and cost-effective method for the quality determination of herbal products of A. racemosus. The developed HPTLC chromatograms would serve as a reference for the quality assessment of commercial products of A. racemosus in future.

Optimization of HPLC-MS/MS method for determination of antimalarial adulterants in herbal products.

The use of herbal products is booming all over the world because of being believed as safer than conventional drugs and free of side effects. However, there are untrustworthy manufacturers who adulterate herbal products by adding conventional drugs which might eventually lead to microbial resistance and herb-to-drug interactions. There is a need to develop methods for detecting adulterants in herbal products. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous identification and determination of conventional antimalarials (chloroquine, quinine, sulfadoxine, pyrimethamine, mefloquine, lumefantrine, amodiaquine, artemisinin, dihydroartemisinin, artesunate and artemether) in herbal products was developed. Stable isotopically labelled compounds (artemether-d3, quindine-d3, and sulfadoxine-d3) were used as internal standards (ISs) for quantitative analysis. Extraction of analytes was performed using methanol: water: formic acid (90:10:0.1, v/v) and chromatographic separation was done in a gradient mode using mobile phase A: Ultrapure water containing 0.1% formic acid and 1mM ammonium formate and mobile phase B: Acetonitrile/methanol (50:50) containing 0.1% formic acid and 1mM ammonium formate. The calibration curves were linear (r2 ≥ 0.991) over the range of 0.001-0.3µgmL-1 for all compounds. The limit of detection (LOD) ranged from 0.002 to 0.02μgmL-1 while the limit of quantification (LOQ) ranged from 0.006 to 0.08μgmL-1. Accuracy, expressed as recovery of spiked herbal products ranged from 52 to 128%. The precision, expressed as percent relative standard deviation (%RSD) at two concentration levels, ranged from 1.0 to 13.8%. The matrix effect expressed as the matrix factor (MF) ranged from 0.77 to 0.97. The developed method was used to identify and quantify conventional antimalarials in herbal product samples from Tanzania. Ten out of 50 herbal products were found to contain amodiaquine, sulfadoxine, pyrimethamine, mefloquine, dihydroartemisinin, artemether and lumefantrine. The developed method is considered a valuable tool for getting a better understanding of the adulteration of conventional antimalarials in herbal products.

Thin Layer Chromatographic Method for Detection of Conventional Drug Adulterants in Herbal Products

Commercially available conventional drugs have been used to adulterate herbal products. Considering the rapid growth of herbal products’ market, it is essential to screen herbal products for the presence of conventional drugs. Simple analytical methods are needed for the rapid screening of conventional drugs that are likely to be adulterated in herbal products. Thin layer chromatography (TLC) methods for screening twelve conventional drugs in herbal products have been developed and applied. The analytes were extracted from herbal products using acetonitrile:methanol:acetic acid:water (4:4:1:1, v/v). Solvent mixture of dichloromethane:ethyl acetate:methanol (75:15:10, v/v) separated well trimethoprim, sildenafil, paracetamol, and sulfamethoxazole while pyrimethamine, metronidazole, and sulfadoxine were well separated by dichloromethane:ethyl acetate:methanol (77.5:12.5:10, v/v). In addition, acetyl salicylic acid, ibuprofen, diclofenac, quinine, and lumefantrine were well separated by ethyl acetate:methanol:30% ammonia (75:22.5:2.5, v/v). Chromatographic separations were found to be highly reproducible, and more than 10 samples can be analysed in one run. The method was applied in the screening of 229 herbal products. Consequently, 24.0% of the samples contained one adulterant, while 21.4% contained at least two adulterants. All conventional drugs detected in herbal products were not mentioned on the labels and therefore the consumers are kept unaware of their side effects and health problems. Further studies for confirming and quantitatively determining the adulterants in a wide range of products as well as a systematic toxicological analysis of the adulterants in herbal products are recommended.

A HPLC-MS/MS method for screening of selected antibiotic adulterants in herbal drugs.

The use of herbal products adulterated with conventional drugs increases the risk of developing microbial resistance and causes herb-to-drug interaction, leading to severe clinical consequences. The complex nature of herbal products has been a challenge for the unambiguous identification of adulterants. The improved analytical selectivity and sensitivity of hyphenated techniques such as high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) enable the confirmatory screening of adulterants in herbal products. Simultaneous screening of adulterants is necessary and efficient because it has been established that more than one chemical adulterant may be present in one herbal product. An HPLC-MS/MS method for the simultaneous detection and quantification of amoxicillin, ampicillin, metronidazole, ciprofloxacin, sulfamethoxazole, and trimethoprim in powdered herbal drugs was developed. Deuterated metronidazole-d3, trimethoprim-d3, ciprofloxacin-d8, and sulfamethoxazole-d4 were used as internal standards (ISs). For each analyte, two transitions were monitored using protonated molecules as precursor ions. The extraction of analytes from herbal products was performed using a simple methanol : water : formic acid (90 : 10 : 0.05, v/v) extraction solvent. Chromatographic separation was done in a gradient of 0.01% formic acid in methanol and 0.01% formic acid in MilliQ water. The calibration curves were linear (r2 ≥ 0.996) over the range of 0.005-2.5 μg mL-1 for all compounds except metronidazole, whose range was 0.005-1 μg mL-1. The limit of detection (LOD) ranged from 0.012 to 0.046 μg mL-1, while the limit of quantification (LOQ) ranged from 0.066 to 0.153 μg mL-1. The accuracy, expressed as the recovery of spiked herbal products, ranged from 45% to 114%. The precision, expressed as relative standard deviation (RSD) at two concentration levels, ranged from 1.6% to 15.9%. The matrix effect expressed as the matrix factor (MF) ranged from 0.79 to 0.92. The developed method was applied to powder herbal products purchased in Tanzania. Amoxicillin, ampicillin, trimethoprim, sulfamethoxazole, and ciprofloxacin were not detected in all samples. Metronidazole was detected in eight samples with the highest concentration of 1.38 μg g-1. The developed method is suitable for the detection of all the studied antibiotic adulterants in herbal products. Quantification can be performed for all the compounds except ciprofloxacin due to its lower recovery.

Development of a molecular marker based on chloroplast gene for specific identification of Korean Hibiscus (Hibiscus syriacus \u2018Simbaek\u2019)

Due to the rise in substitution and adulteration of herbal products, as well as the lack of genetic information on Hibiscus plants, more molecular markers are needed to understand the genetic diversity and avoid their misidentification. There are many allelic variants of the functional genes in Hibiscus and other plants, which control their respective phenotypes and other characteristics. Identifying alleles of the desired trait by determining diversification through gene-typing allele-specific markers for authentication is, therefore, a potent strategy. The purpose of this study was to use insertion/deletion (InDel) markers to identify Hibiscus syriacus cultivars. We developed a novel InDel marker for Korean Hibiscus ‘Simbaek’, based on the trnL-F region of the chloroplast gene. Through this InDel site, a modified specific primer pair and a novel multiplex polymerase chain reaction (PCR) system were developed for specific identification of the Korean Hibiscus Simbaek cultivar. The molecular markers developed in this study were highly specific and accurately authenticated as Simbaek from the five main cultivars of H. syriacus. Taken together, the described method is a potential tool for the identification and selection of germplasm resource of Simbaek cultivar.Graphical

Review of Adulterated Herbal Products and Supplements and Methods of Analysis

Herbal products and supplements are widely used for primary health care. Many reports of drugs added to these products, such as weight-loss supplements, analgesic herbal products, and erectile dysfunction supplements. This study aims to find out products that are often mixed with drugs and effective methods used for their analysis. The method used in this research is a systematic review. The resources were retrieved through searching the keywords related to the world adulterated herbal products and supplements in the available databases and search engines including Science Direct, PubMed, Springerlink, and Google Scholar. Based on PRISMA flow diagram, a systematic review showed that sibutramine HCl, sildenafil citrate, dexamethasone, and sodium diclofenac are most commonly found in the herbal and supplement products and the ESI-MS method is an effective method for their analysis.

The DNA-Based Authentication of Commercial Herbal Products Reveals Their Globally Widespread Adulteration.

The herbal products, sold worldwide as medicines or foods, are perceived as low risk because they are considered natural and thus safe. The quality of these products is ineffectively regulated and controlled. The growing evidence for their lack of authenticity is causing deep concern, but the scale of this phenomenon at the global, continental or national scale remains unknown. We analyzed data reporting the authenticity, as detected with DNA-based methods, of 5,957 commercial herbal products sold in 37 countries, distributed in all six inhabited continents. Our global survey shows that a substantial proportion (27%) of the herbal products commercialized in the global marketplace is adulterated when their content was tested against their labeled, claimed ingredient species. The adulterated herbal products are distributed across all continents and regions. The proportion of adulterated products varies significantly among continents, being highest in Australia (79%), South America (67%), lower in Europe (47%), North America (33%), Africa (27%) and the lowest in Asia (23%). The commercial HPs’ authenticity among the 37 countries included in our global analysis ranges between 0 and 100% from the total number of product reported for each specific national marketplace. For 9 countries, more than 100 products were successfully DNA-based authenticated and reported. From these countries, the highest percentage of adulterated commercial HPs was reported for Brazil (68%), followed distantly by Taiwan (32%), India (31%), USA (29%), followed closely by Malaysia (24%), Japan (23%), South Korea (23%), Thailand (20%), and China (19%). Our results confirm the large-scale presence of adulterated herbal products throughout the global market. The adulterated herbal products contain undeclared contaminant, substitute, and filler species, or none of the labeled species, which all may be accidental or intentional, economically-motivated and fraudulent. Due to the ever-increasing analytical sensitivity of the high throughput DNA sequencing, increasingly used for the untargeted, simultaneous multi-taxa identification, the proportion of adulterated HPs detected on the global market is expected to increase. In the context of the increasing demand for HPs, the limited supply of raw materials derived from many plant species, some of which being already nationally or internationally protected and having various degrees of trade restrictions, adds up to the differences and discrepancies between national HPs’ regulatory frameworks and further increases the risks of adulteration of many types of herbal products. The globally widespread adulteration is a serious threat to consumers’ well-being and safety, in spite of herbal products’ claimed or expected health benefits.

Herbal Medicinal Products in Turkey (Standardization, Production and Adulteration of Herbal Products in Turkey)

Fitoterapinin dünyadaki tarihsel gelişimi birçok çalışmada defalarca vurgulanmıştır. Ülkemizde ise getat mevzuatıyla beraber popülaritesi artan bir bilim dalı haline gelmiştir. Fitoterapi eğitimleri yıllardır Eczacılık Fakültelerinde Farmakognozi Anabilim dalı adı altında verilmekteydi. T.C Sağlık Bakanlığı’nın 2014 Geleneksel ve Tamamlayıcı Tıp (GETAT) mevzuatıyla beraber, yetki verilen üniversitelerce eğitimler yaygınlaştırılmış, eğitim alan hekim ve eczacı sayısı hızla artmıştır. Sonuçta hedef insan sağlığı ve tedavi olunca, fitoterapinin de gelişigüzel uygulanması düşünülemeyeceğinden, kanıta dayalı fitoterapi konusu gündeme gelmiştir ki; bunu da doğru endikasyonda, uygun dozda istenen maximum etki ve her uygulamada aynı etki şeklinde açıklamak doğru olacaktır. Bu noktada kullanılacak bitkisel ürünlerde kalite ve standardizasyonun önemi iyice belirginleşmektedir. Bu nedenle bu çalışmada bitkinin yetiştirilme sürecinden, drog ve ilaç haline gelinceye ve de artık uygulama ve tedavi boyutunda olması gereken kalite ve standardizasyon konusuna vurgu yapılması amaçlanmıştır.

Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis.

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.

Survey of traditional Dai medicine reveals species confusion and potential safety concerns: a case study on Radix Clerodendri Japonicum

The adulteration of herbal products is a threat to consumer safety. In the present study, we surveyed the species composition of commercial Radix Clerodendri Japonicum products using DNA barcoding as a supervisory method. A reference database for plant-material DNA-barcode was successfully constructed with 48 voucher samples from 12 Clerodendrum species. The database was used to identify 27 Radix Clerodendri Japonicum decoction piece samples purchased from drug stores and hospitals. The DNA sequencing results revealed that only 1 decoction piece (3.70%) was authentic C. japonicum, as recorded in the Dai Pharmacopeia, whereas the other samples were all adulterants, indicating a potential safety issue. The results indicate that decoction pieces that are available in the market have complex origins and that DNA barcoding is a suitable tool for regulation of Dai medicines.

Steroids in traditional Chinese medicine: what is the evidence?

Local healthcare providers often question the possible steroidal activity of traditional Chinese medicine (TCM) herbs or herbal products and implicate them as a cause for adrenal insufficiency or Cushing's syndrome in patients with a history of TCM intake. We conducted a comprehensive database search for evidence of potential glucocorticoid, mineralocorticoid, androgenic or oestrogenic activity of herbs or herbal products. Overall, there are not many herbs whose steroidal activity is well established; among these, most cases were based on preclinical studies. Liquorice root may cause pseudoaldosteronism through interference with the steroidogenesis pathway. Although ginseng and cordyceps have some in vitro glucocorticoid activities, the corroborating clinical data is lacking. Deer musk and deer antler contain androgenic steroids, while epimedium has oestrogenic activity. On the other hand, adulteration of herbal products with exogenous glucocorticoids is a recurrent problem encountered locally in illegal products masquerading as TCM. Healthcare providers should stay vigilant and report any suspicion to the relevant authorities for further investigations.

Isolation and identification of a novel sildenafil analogue adulterant in herbal products

ABSTRACTA novel sildenafil analogue found in herbal products by a routine drug-adulteration screening programme was isolated by column chromatography. On the basis of extensive 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy and mass spectral analysis, the structure of a new compound YJ-07 was established as 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl) benzenesulfonamide. It common name is aminosildenafil.

Authentication of the botanical origin of Western herbal products using Cimicifuga and Vitex products as examples.

Various herbal medicines have been developed and used in various parts of the world for thousands of years. Although locally grown indigenous plants were originally used for traditional herbal preparations, Western herbal products are now becoming popular in Japan with the increasing interest in health. At the same time, there are growing concerns about the substitution of ingredients and adulteration of herbal products, highlighting the need for the authentication of the origin of plants used in herbal products. This review describes studies on Cimicifuga and Vitex products developed in Europe and Japan, focusing on establishing analytical methods to evaluate the origins of material plants and finished products. These methods include a polymerase chain reaction-restriction fragment length polymorphism method and a multiplex amplification refractory mutation system method. A genome-based authentication method and liquid chromatography-mass spectrometry-based authentication for black cohosh products, and the identification of two characteristic diterpenes of agnus castus fruit and a shrub chaste tree fruit-specific triterpene derivative are also described.Electronic supplementary materialThe online version of this article (doi:10.1007/s11418-016-1006-0) contains supplementary material, which is available to authorized users.

The Potential Power of Bar-HRM Technology in Herbal Medicine Identification.

The substitution of low-cost or adulterated herbal products for high-priced herbs makes it important to be able to identify and trace herbal plant species and their processed products in the drug supply chain. PCR-based methods play an increasing role in monitoring the safety of herbal medicines by detecting adulteration. Recent studies have shown the potential of DNA barcoding combined with high resolution melting (Bar-HRM) analysis in herbal medicine identification. This method involves precisely monitoring the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by a gradual increase in temperature. Since the melting profile depends on the GC content, length, and strand complementarity of the amplification product, Bar-HRM analysis opens up the possibility of detecting single-base variants or species-specific differences in a short region of DNA. This review summarizes key factors affecting Bar-HRM analysis and describes how Bar-HRM is performed. We then discuss advances in Bar-HRM analysis of medicinal plant ingredients (herbal materia medica) as a contribution toward safe and effective herbal medicines.

Two-dimensional correlation infrared spectroscopy applied to the identification of ephedrine and pseudoephedrine in illegally adulterated slimming herbal products.

Two-dimensional correlation spectroscopy (2DCOS) was employed for the identification of ephedrine (Ep) and pseudoephedrine (Ps) present in illegally adulterated slimming herbal products (SHPs). Second derivative (SD) spectral pretreatment was used prior to 2DCOS analysis to highlight specific features not readily observable by Fourier transform infrared spectroscopy (FTIR), SD-FTIR, or original 2DCOS, leading to enhanced resolution and a reduced lower limit of detection (<1% in this study). After examining the power spectra of suspicious SHPs, bands containing characteristic peaks for Ep (701, 747, 1042, 1363, 1375, 1451, 1478 cm-1 etc) and/or Ps (703, 767, 1037, 1375, 1428, 1455, 1590 cm- 1, etc.) were selected to construct synchronous and asynchronous maps for further analysis, while the latter was applied to discriminate positive SHPs adulterated simultaneously with Ep and Ps. The proposed method is simple and economical and has the potential to identify other chemicals in illegally adulterated herbal products. Copyright © 2016 John Wiley & Sons, Ltd.

Survey of commercial Rhodiola products revealed species diversity and potential safety issues.

The adulteration of herbal products is a threat to consumer safety. Here we surveyed the species composition of commercial Rhodiola products using DNA barcoding as a supervisory method. A Rhodiola dietary supplement DNA barcode database was successfully constructed using 82 voucher samples from 10 Rhodiola species. Based on the DNA barcoding standard operating procedure (SOP), we used this database to identify 100 Rhodiolae Crenulatae Radix et Rhizoma decoction piece samples that were purchased from drug stores and hospitals. The results showed that only 36 decoction piece sequences (40%) were authentic R. crenulata, which is recorded in Chinese Pharmacopeia, whereas the other samples were all adulterants and may indicate a potential safety issue. Among the adulterants, 35 sequences (38.9%) were authenticated as R. serrata, nine sequences (10%) were authenticated as R. rosea, which is documented in the United States Pharmacopeia, and the remaining samples were authenticated as other three Rhodiola species. This result indicates decoction pieces that are available in the market have complex origins and DNA barcoding is a convenient tool for market supervision.

Identification and screening of a tadalafil analogue found in adulterated herbal products

A tadalafil analogue was detected in an herbal product by high performance liquid chromatography-diode array detector (HPLC-DAD) with a similar chromatographic retention time to tadalafil. The compounds were separated using semi-preparative HPLC. The structure of the detected tadalafil analogue was elucidated by LC-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) and nuclear magnetic resonance (NMR) spectroscopy. A positive ion at m/z 404.1644 was detected by LC-Q-TOF/MS, corresponding to a molecular formula of C23H22N3O4. This unknown compound was identified as an analogue of tadalafil containing an additional methylene group and named homotadalafil. Homotadalafil was detected in 10 of 91 herbal products at concentrations of 0.058mgg−1 to 8.735mgg−1.

Metabolites of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identified in human urine and liver microsomal preparations using liquid chromatography high-resolution mass spectrometry.

New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N-​(1-Adamant​yl)-​1-​(5-​fluoropentyl)-​1H-​indazole-​3-​carboxamide (5F-AKB-48), also known as 5F-APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW-type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high-resolution accurate-mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase-I metabolites of 5F-AKB-48. The modifications included mono-, di-, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N-fluoropentylindazole moiety, dealkylation of the N-fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time-dependent formation of mono-, di-, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F-AKB-48 for use in clinical and forensic screening.