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Seven Years of Third-Party Quality Data on a Commercial Tongkat Ali (Eurycoma longifolia) Root Extract Produced by Ultrasound-Assisted Enzymatic Extraction: Marker Content, Contaminants and Conformity with MS 2409:2011

Submitted:

24 October 2026

Posted:

27 October 2026

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Abstract
Market surveys of Tongkat Ali (Eurycoma longifolia) products have repeatedly found heavy-metal contamination and inconsistent marker content, yet manufacturers rarely publish their own quality data. This communication reports, retrospectively, fourteen third-party analytical reports issued between 2018 and 2025 for a commercial root extract produced by patented ultrasound-assisted enzymatic extraction, in which ultrasonic cavitation is combined with enzymatic cell-wall hydrolysis. Two samples contained 2.71% and 7.51% eurycomanone by ultra-performance liquid chromatography, 1.8 to 9.4 times the range specified in Malaysian Standard MS 2409:2011. Lead, arsenic, cadmium and mercury were not detected or were at most 17% of ASEAN limits for health supplements in all five lots tested. Aflatoxins were not detected, and none of 255 pesticides was detected in the root raw material. Salmonella and indicator organisms were absent or below reporting limits throughout, while aerobic counts fell from 6.0 × 10⁴–2.5 × 10⁵ to 1.1–9.2 × 10² colony-forming units per gram after 2021. Protein (10.0 g/100 g) was below the MS 2409:2011 minimum of 22%, and a froth test did not detect saponins. Because no conventional extract was analysed in parallel, these data establish safety and marker conformity but not process superiority; a controlled comparison is proposed.
Keywords: 
Eurycoma longifolia
;  
Tongkat Ali
;  
eurycomanone
;  
ultrasound-assisted extraction
;  
ultrasonic cavitation
;  
enzyme-assisted extraction
;  
quality control
;  
heavy metals
;  
microbiological quality
;  
MS 2409
Subject: 
Biology and Life Sciences  -   Food Science and Technology

1. Introduction

Tongkat Ali (Eurycoma longifolia Jack, Simaroubaceae) is among the most widely consumed medicinal plants of Southeast Asia, and its root extract is now sold as a food ingredient, a traditional product and a health supplement [1,2]. The quassinoid eurycomanone is the principal marker of the root and the compound most often used to standardise extracts, although it is also the most cytotoxic of the major quassinoids [3,4]. Malaysian Standard MS 2409:2011 specifies a freeze-dried water extract containing 0.8 to 1.5% eurycomanone together with minimum protein, polysaccharide and glycosaponin fractions [5,6], and most clinical trials have used extracts made to that standard [7].
Quality failures in this product category are well documented. In random samples of 100 products on the Malaysian market, 8% exceeded the national limit of 10 ppm for lead [8] and 26% exceeded the 0.5 ppm limit for mercury [9]. Chromatographic and metabolomic fingerprinting has shown that commercial products differ widely in composition from authentic root [10,11], and adulteration of botanical ingredients is common across markets [12]. Eurycomanone content also varies with the origin of the root [13]. Against this background, published data from manufacturers on their own extracts would be useful to regulators and formulators, but are scarce.
Ultrasound-assisted extraction works mainly through acoustic cavitation: the formation and violent collapse of microbubbles in the extraction medium, which generates local shear forces, microjets and shock waves that fragment cell walls, erode the tissue surface and speed up mass transfer of solutes into the medium [14,15]. These effects allow extraction at lower temperatures and in shorter times than conventional maceration or decoction, which is relevant for heat-sensitive constituents [15]. Dense, lignified materials such as woody roots are among those that benefit, because the solvent otherwise penetrates them slowly. Enzyme-assisted extraction acts on the same barrier by hydrolysing cellulose, hemicellulose and pectin in the cell wall [16,17], and recent work indicates that combining ultrasound with enzymatic treatment can be synergistic, depending on treatment sequence and the complexity of the biomass [18]. Ultrasound can also degrade sensitive compounds if poorly controlled [19], so the composition of an extract made this way cannot be assumed to match a conventional water extract. Extraction methods applied specifically to E. longifolia have been reviewed [20], but no multi-year quality dataset for an extract made by combined enzymatic and ultrasonic processing has been published.
This communication therefore reports all the third-party analytical data held by one manufacturer for a commercial E. longifolia root extract produced by patented ultrasound-assisted enzymatic extraction, and compares them with MS 2409:2011, ASEAN contaminant limits and the published literature. It is a retrospective record of routine quality testing. It does not compare the process with conventional extraction and makes no claim about efficacy.

2. Materials and Methods

2.1. Extract and Data Source

The extract was produced from E. longifolia roots by Bionutricia Manufacturing Sdn Bhd (Selangor, Malaysia) using the company’s ultrasound-assisted enzymatic extraction process, which is protected by Malaysian patent MY-188945-A. The process combines an ultrasonic extraction stage with enzymatic treatment of the milled root. Ultrasonic and enzymatic conditions are proprietary and are not reported. The extract is marketed in grades standardised to eurycomanone.
The company’s electronic document archive was searched for external analytical reports on Tongkat Ali extract or its root raw material issued between 2018 and 2025. Fourteen reports from four laboratories were found, and all were included; none was excluded on the basis of its result (Table 1). Internal certificates of analysis that stated conformity without reporting measured values were not used as data.

2.2. Analytical Methods

All analyses were performed by the issuing laboratories on samples submitted by the manufacturer, using the methods stated on each report. Eurycomanone was quantified by the Forest Research Institute Malaysia (FRIM) using ultra-performance liquid chromatography on a BEH C18 column with ultraviolet detection at 244 nm. Qualitative phytochemical screening at FRIM followed conventional precipitation, colour and froth tests on 100 mg of sample [21]. Total phenolic content was determined by UKM Unipeq using the Folin–Ciocalteu method and expressed as gallic acid equivalents [22]. Proximate composition, minerals and vitamin C were determined by ALS Technichem using the AOAC-based methods listed in Table 3. Heavy metals were determined by SGS (Malaysia) by inductively coupled plasma optical emission spectrometry (2018 to 2022) or mass spectrometry (2025), and mercury by direct mercury analysis or inductively coupled plasma mass spectrometry. Microbiological counts followed AOAC or ISO methods as stated on each report. Aflatoxins B1, B2, G1 and G2 were determined by high-performance liquid chromatography with fluorescence detection with reference to ISO 16050:2003 and AOAC 999.07, and pesticide residues in the root raw material by liquid chromatography–tandem mass spectrometry with reference to EN 15662 [23]. ALS Technichem and SGS (Malaysia) state accreditation under Standards Malaysia on their reports.

2.3. Benchmarks and Data Treatment

Composition was compared with MS 2409:2011 [5,6]. Heavy metals and microbiological counts were compared with the ASEAN guidelines on limits of contaminants for health supplements [24], and aflatoxin B1 with the European Pharmacopoeia limit for herbal drugs of 2 µg/kg [25]. Results are reported as issued. Values below the reporting limit are given as not detected (ND) with the limit in brackets. Daily intake of eurycomanone and of quantified metals was estimated for 200 mg and 400 mg of extract per day, the range of doses used in most clinical trials of standardised extracts [7]. No statistical comparison was made, because only one result was available for each lot and parameter.

3. Results

3.1. Eurycomanone

The two samples analysed by FRIM in October 2018 contained 2.71% and 7.51% eurycomanone (Table 2). The laboratory reported a relative standard deviation of 0.36% for the first sample. Both values exceed the MS 2409:2011 range of 0.8 to 1.5%. At a serving of 200 mg, the 2.71% material supplies 5.4 mg of eurycomanone, and the 7.51% material 15.0 mg, compared with 1.6 to 3.0 mg from an extract made to the standard. The 7.51% sample lies between a standardised water extract and the quassinoid-rich fraction described by Low et al., which contained 18.5% eurycomanone [26].
Table 2. Eurycomanone content of two extract samples compared with the MS 2409:2011 specification for a standardised Tongkat Ali water extract [5,6] and with a quassinoid-rich fraction [26]. Eurycomanone delivered per 200 mg serving is calculated from the measured content.
Table 2. Eurycomanone content of two extract samples compared with the MS 2409:2011 specification for a standardised Tongkat Ali water extract [5,6] and with a quassinoid-rich fraction [26]. Eurycomanone delivered per 200 mg serving is calculated from the measured content.
Material Eurycomanone (% w/w) Per 200 mg serving (mg) Ratio to MS 2409 range
Sample JN 107/18 (this work; RSD 0.36%) 2.71 5.4 1.8 to 3.4
Sample JN 109/18 (this work) 7.51 15.0 5.0 to 9.4
MS 2409:2011 water extract specification 0.8 to 1.5 1.6 to 3.0 1
Quassinoid-rich fraction (Fr 2) 18.5 37.0 12 to 23

3.2. Composition and Phytochemical Screening

The 2–3% grade analysed in 2021 consisted mainly of carbohydrate (66.4 g/100 g, of which 12.6 g was dietary fibre), with 17.5 g/100 g ash, 10.0 g/100 g protein and negligible fat (Table 3). The ash was rich in potassium (3610 mg/100 g) and calcium (1280 mg/100 g). Phytochemical screening of a liquid sample detected weak reactions for flavonoids and triterpenes and did not detect alkaloids, tannins, steroids or foam-forming saponins. Total phenolic content was 3.79 mg gallic acid equivalents per 100 g.
Table 3. Composition and phytochemical screening results. Proximate values are for the 2–3% eurycomanone grade (ALS, May 2021); total phenolic content and screening are from the December 2018 samples. The MS 2409:2011 values are those published for a conforming product [6].
Table 3. Composition and phytochemical screening results. Proximate values are for the 2–3% eurycomanone grade (ALS, May 2021); total phenolic content and screening are from the December 2018 samples. The MS 2409:2011 values are those published for a conforming product [6].
Parameter Result Method MS 2409:2011 or comment
Protein (g/100 g) 10.0 Kjeldahl, AOAC 972.43 At least 22
Total carbohydrate (g/100 g) 66.4 By difference Polysaccharide at least 30 (method differs)
Total dietary fibre (g/100 g) 12.6 AOAC 985.29 Not specified
Ash (g/100 g) 17.5 Gravimetric Not specified
Moisture (g/100 g) 6.1 Moisture analyser Not specified
Fat (g/100 g) Below 0.1 Gravimetric Not specified
Calcium; potassium; sodium; iron (mg/100 g) 1280; 3610; 167; 3.97 ICP-OES, AOAC 984.27 Not specified
Vitamin C (mg/100 g) 27 AOAC 967.21 Not specified
Energy (kcal/100 g) 306 Calculated Not specified
Total phenolic content (mg gallic acid equivalents/100 g) 3.79 Folin–Ciocalteu [22] Not specified
Alkaloids; tannins; steroids (screening) Not detected Colour and precipitation tests [21] Consistent with quassinoid-dominated root
Saponins (froth test) Not detected Froth test Glycosaponin at least 40 (gravimetric fraction)
Flavonoids; triterpenes (screening) Detected, weak (1+) Colour tests [21] Quassinoids are degraded triterpenoids

3.3. Heavy Metals

Lead, cadmium, arsenic and mercury were not detected in the four lots tested from 2018 to 2022 (Table 4). In the 2025 lot, analysed by the more sensitive mass-spectrometric method, lead was quantified at 0.71 mg/kg, arsenic at 0.15 mg/kg, cadmium at 0.05 mg/kg and antimony at 0.28 mg/kg, while mercury remained below 0.02 mg/kg. The highest value relative to its ASEAN limit was lead, at 7% of the limit; cadmium was at 17%. At 400 mg of extract per day, the 2025 lot would supply about 0.28 µg of lead, 0.06 µg of arsenic and 0.02 µg of cadmium daily.

3.4. Microbiological Quality

Salmonella was absent from 25 g, and coliforms, Escherichia coli, Staphylococcus aureus, yeasts and moulds were below their reporting limits, in every lot in which they were tested (Table 5). Aerobic plate counts in the four results from 2018 to 2021 ranged from 6.0 × 10⁴ to 2.5 × 10⁵ colony-forming units per gram. These counts are within the ASEAN category C limit of 5 × 10⁵ but above the category B limit of 5 × 10⁴. The three results from 2022 and 2025 ranged from 1.1 × 10² to 9.2 × 10² colony-forming units per gram, about two to three orders of magnitude lower and within category B.

3.5. Aflatoxins and Pesticide Residues

Aflatoxins B1, B2, G1 and G2 and their sum were each below 0.5 µg/kg in the 2022 lot, less than a quarter of the European Pharmacopoeia limit of 2 µg/kg for aflatoxin B1 [25]. None of 255 pesticide residues screened in the 2022 root raw material was detected, at reporting limits of 0.01 to 0.05 mg/kg.

4. Discussion

4.1. Contaminants

Across seven years and up to five lots per parameter, the extract met every ASEAN contaminant limit for health supplements, with the largest value at 17% of its limit. This contrasts with market surveys in which up to a quarter of Tongkat Ali products exceeded Malaysian limits for mercury or lead [8,9]. The comparison should not be over-read: the surveys sampled finished products from many sources, whereas these results come from a single producer’s own extract. The quantification of lead, arsenic and cadmium in 2025 after four years of non-detection most likely reflects the change from optical-emission to mass-spectrometric detection, whose lower reporting limits revealed trace levels that were previously below 0.5 mg/kg; the 2018 and 2019 lead results, with a reporting limit of 0.01 mg/kg, show that lead content does vary between lots. Pesticide residues were tested only in the root, and aflatoxins only once, so these results indicate the absence of a problem rather than establish lot-to-lot consistency.
The fall in aerobic plate count after 2021 is the clearest trend in the data. A reduction of two to three orders of magnitude moved the product from ASEAN category C to the stricter category B. These records do not include information on process, hygiene or raw-material changes over this period, so the reason for the fall cannot be established from the data and should not be attributed to the extraction process itself.

4.2. Marker Content

Both eurycomanone values were above the MS 2409:2011 range. This matters for three reasons. First, products are often labelled by extract weight, so a consumer taking 200 mg of a 2.71% extract receives 1.8 to 3.4 times the eurycomanone of the same weight of a standard extract, and a consumer taking the 7.51% material 5.0 to 9.4 times as much (Table 2). Second, eurycomanone is the most cytotoxic of the major quassinoids [3], and safety studies of the quassinoid-rich fraction were conducted on a material with 18.5% eurycomanone [4,26], so safety data for standard water extracts may not transfer directly to higher-marker extracts. Third, eurycomanone is poorly absorbed whether given pure or within a water extract [27], so a higher marker content does not necessarily mean higher systemic exposure. We therefore suggest that labels state eurycomanone in milligrams per serving, not only as a percentage.
A higher marker content is consistent with more efficient release of quassinoids when cavitation disrupts the woody root tissue and enzymes hydrolyse the cell wall [14,15,16,18]. Other explanations are also possible. Eurycomanone content varies with the provenance of the root, with reported values up to 1.46% between Malaysian sources [13,28]. Root age is widely held among growers and processors to raise eurycomanone content, with older roots preferred, but we found no published study that has measured this relationship, and the age of the roots used for these samples was not recorded. The difference could also reflect the grade being made or post-extraction concentration. The origin of the 7.51% sample relative to commercial grades is not documented in the archive. These data therefore cannot show that the process increases eurycomanone yield.

4.3. Conformity with Other MS 2409:2011 Criteria

The extract did not meet two other compositional expectations of MS 2409:2011. Protein was 10.0 g/100 g, against a minimum of 22%. The Kjeldahl method measures total nitrogen, so enzymatic hydrolysis of proteins to peptides would not by itself lower the result; the shortfall is more likely to reflect a different balance of extracted carbohydrate and mineral matter, as the high ash and potassium contents suggest. The froth test did not detect saponins, whereas MS 2409:2011 requires at least 40% glycosaponin. The two results are not directly comparable: glycosaponin in the standard is a gravimetric fraction, while the froth test detects only foam-forming saponins and was applied to a liquid sample. Saponins in E. longifolia water extract are recovered by solvent fractionation and are present as glycosylated compounds that may not foam strongly [29]. MS 2409:2011 was written for a freeze-dried water extract, and an extract made by a different process need not conform to it, but the differences should be stated on specifications rather than implied by citing the standard.
The total phenolic content of 3.79 mg gallic acid equivalents per 100 g is very low and agrees with the weak flavonoid and absent tannin reactions in screening. E. longifolia root is not a phenolic-rich material, and its activity is usually attributed to quassinoids, polysaccharides and peptide fractions [1,30]. Antioxidant claims based on phenolic content are therefore not supported for this extract. The sample form for this test was not recorded, so dilution in a liquid sample may have contributed to the low value.

4.4. Limitations

This is a retrospective compilation of routine reports and has the limitations of that design. Each parameter was measured once per lot, without replicate lots or measures of between-lot variance, and different parameters were measured on different lots. Eurycomanone was measured in only two samples, both in 2018, and has not been re-measured by an independent laboratory since. The laboratories analysed samples submitted by the manufacturer and did not perform the sampling. Several methods changed over the period, notably for heavy metals. No conventional water extract from the same root was analysed in parallel, so no inference about the relative performance of the process is possible. Process conditions are not disclosed, which limits reproducibility. The author owns the manufacturer, which also owns the process patent.

4.5. A Controlled Comparison

The question this dataset cannot answer, whether ultrasound-assisted enzymatic extraction yields a better extract than conventional hot-water extraction, requires a prospective study. A suitable design would divide a single authenticated lot of milled root into portions extracted in triplicate by the patented process, by hot-water extraction under MS 2409:2011 conditions, and by ultrasound alone and enzyme alone, using roots of recorded age and provenance and with drying conditions held constant. Including an ultrasound-only arm would show how much of any gain is due to cavitation itself. Outcomes would include extraction yield, eurycomanone and at least three other quassinoids by validated chromatography, protein, polysaccharide and glycosaponin by the methods of MS 2409:2011, a chromatographic fingerprint [10], contaminants, and eurycomanone stability over 12 months. Results should be analysed by an independent laboratory blinded to treatment, with yields expressed per gram of root as well as per gram of extract, since concentration alone can raise marker content without improving extraction.

5. Conclusions

Fourteen third-party reports spanning 2018 to 2025 show that a commercial E. longifolia root extract made by patented ultrasound-assisted enzymatic extraction met ASEAN limits for heavy metals and pathogens, with no detected aflatoxins or pesticide residues, and contained eurycomanone at 2.71 to 7.51%, above the MS 2409:2011 range. Aerobic counts fell by two to three orders of magnitude after 2021. The extract did not meet the protein or glycosaponin criteria of MS 2409:2011 and was low in phenolics. These data support the safety and marker conformity of the extract, but not a claim that the process is superior to conventional extraction; that requires the controlled comparison outlined above. Manufacturers publishing complete quality records, including unfavourable results, would help establish a quality baseline for this widely used botanical.

Author Contributions

Kuak Ping Ng conceived the study, compiled and interpreted the analytical data, and wrote the manuscript.

Funding

This research received no external funding. The analytical tests were commissioned and paid for by Bionutricia Manufacturing Sdn Bhd as part of routine quality control.

Conflicts of interest

The author is the owner of the Bionutricia group, which manufactures and sells the extract described in this communication and owns Malaysian patent MY-188945-A covering the extraction process. The analytical laboratories had no role in the design of this communication, the interpretation of the data or the decision to publish.

Use of artificial intelligence

An artificial intelligence assistant (Claude, Anthropic) was used to support data compilation, literature searching, drafting and language editing. The author reviewed, edited and verified all content, values and references and takes full responsibility for the manuscript.

Data availability

All data supporting the findings of this study are contained within the article (Table 1, Table 2, Table 3, Table 4 and Table 5).

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Table 1. Third-party analytical reports included in this communication, in date order. All reports were issued to the manufacturer for single submitted samples; sampling was not performed by the laboratories.
Table 1. Third-party analytical reports included in this communication, in date order. All reports were issued to the manufacturer for single submitted samples; sampling was not performed by the laboratories.
Report date Laboratory and report Sample as identified on report Parameters
23 Oct 2018 Forest Research Institute Malaysia (FRIM), JN 107/18 Tongkat Ali extract (laboratory ref. 3801018) Eurycomanone (UPLC–UV)
24 Oct 2018 SGS (Malaysia), AE66015 Tongkat Ali extract Heavy metals; microbiology
25 Oct 2018 FRIM, JN 109/18 Tongkat Ali extract (laboratory ref. 3821018) Eurycomanone (UPLC–UV)
1 Nov 2018 ALS Technichem, MF1817318 Tongkat Ali extract Total plate count
17 Dec 2018 FRIM, JN 148/18 Tongkat Ali extract, liquid form Qualitative phytochemical screening
18 Dec 2018 UKM Unipeq, ULUKM/10500/18 Tongkat Ali extract Total phenolic content
13 Aug 2019 SGS (Malaysia), AE88554 Tongkat Ali extract, powder Heavy metals; microbiology
24 Mar 2021 SGS (Malaysia), AF38330 Tongkat Ali extract powder Heavy metals; microbiology
12 May 2021 ALS Technichem, MF2109592 Tongkat Ali extract powder, 2–3% grade Proximate composition; minerals; vitamin C
25 Apr 2022 SGS (Malaysia), AF74809 Tongkat Ali root (raw material) Multiresidue pesticide screen (255 analytes)
28 Apr 2022 SGS (Malaysia), AF74810 Tongkat Ali extract powder Aflatoxins B1, B2, G1, G2
11 Jun 2022 SGS (Malaysia), AF79894 Tongkat Ali extract powder Aerobic plate count
26 Aug 2022 SGS (Malaysia), AF87159 Tongkat Ali extract powder, batch 0722001 Heavy metals; microbiology
26 May 2025 SGS (Malaysia), AH28364 Tongkat Ali extract powder Heavy metals (ICP-MS); microbiology
Table 4. Heavy metals in five lots tested between 2018 and 2025 (mg/kg), compared with the ASEAN limits for health supplements [24]. ND, not detected, with the reporting limit in brackets. ICP-OES was used to 2022 and ICP-MS in 2025.
Table 4. Heavy metals in five lots tested between 2018 and 2025 (mg/kg), compared with the ASEAN limits for health supplements [24]. ND, not detected, with the reporting limit in brackets. ICP-OES was used to 2022 and ICP-MS in 2025.
Metal Oct 2018 Aug 2019 Mar 2021 Aug 2022 May 2025 (limit)
Lead ND (0.01) ND (0.01) ND (0.5) ND (0.5) 0.71 (10.0)
Arsenic ND (0.5) ND (0.5) ND (0.5) ND (0.5) 0.15 (5.0)
Cadmium ND (0.01) ND (0.01) ND (0.5) ND (0.5) 0.05 (0.3)
Mercury ND (0.02) ND (0.02) ND (0.02) ND (0.02) ND (0.02) (0.5)
Antimony ND (0.5) ND (0.5) ND (0.5) ND (0.5) 0.28 (none)
Table 5. Microbiological results for seven extract lots (colony-forming units per gram unless stated), with the ASEAN limits for plant-derived health supplements in categories B and C [24]. ND, not detected, with the reporting limit in brackets.
Table 5. Microbiological results for seven extract lots (colony-forming units per gram unless stated), with the ASEAN limits for plant-derived health supplements in categories B and C [24]. ND, not detected, with the reporting limit in brackets.
Report Aerobic plate count Coliforms E. coli S. aureus Yeasts and moulds Salmonella(25 g)
Oct 2018 (SGS) 2.5 × 10⁵ ND (10) ND (10) ND (10) ND (10) Absent
Nov 2018 (ALS) 6.0 × 10⁴ – – – – –
Aug 2019 6.8 × 10⁴ ND (10) ND (10) ND (10) ND (10) Absent
Mar 2021 1.2 × 10⁵ ND (10) ND (10) ND (10) ND (10) Absent
Jun 2022 9.2 × 10² – – – – –
Aug 2022 1.1 × 10² ND (10) ND (10) ND (10) ND (10) Absent
May 2025 1.4 × 10² ND (3 MPN/g) ND (3 MPN/g) ND (10) ND (10) Absent
Limit, category B 5 × 10⁴ – Absent in 1 g – 5 × 10² Absent
Limit, category C 5 × 10⁵ – Absent in 1 g – 5 × 10⁴ Absent
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