HPLC and ICP-MS: laboratory methods for kratom analysis

In brief

The two reference methods for kratom analysis in accredited laboratories are HPLC (high-performance liquid chromatography) for determining alkaloids and ICP-MS (inductively coupled plasma mass spectrometry) for determining heavy metals. Both methods are described in USP and ISO standards as standard procedures.

HPLC for alkaloids

HPLC allows the separation of individual alkaloids and their quantification. For kratom the standard target is the determination of mitragynine and 7-hydroxymitragynine; extended methods also cover paynantheine, speciogynine and speciociliatine.

Detector

• HPLC-DAD (diode array detector, UV-VIS) - simpler, cheaper, suitable for the routine determination of mitragynine (LOQ of the order of 0.01 %).
• HPLC-MS/MS (tandem mass spectrometry) - orders of magnitude more sensitive, identification based on the mass spectrum, suitable for trace alkaloids including 7-OH-MG (LOQ a few ng/g).

Stationary and mobile phases

A reversed phase is used as standard (C18 column, 100-150 mm × 2.1-4.6 mm, particles 1.7-3.5 μm). The mobile phase is an acetonitrile/water gradient with the addition of formic acid or ammonium formate to adjust the pH.

Sample preparation

Standard procedure: a sample of 0.1-0.5 g of dried powder, extraction into methanol or a methanol/water mixture using ultrasound, filtration (0.22 μm), injection of 1-10 μl. For extracts, the dilution is adjusted to the expected content.

ICP-MS for heavy metals

ICP-MS is the reference method for determining trace concentrations of elements. The principle is the ionisation of the sample in an argon plasma at ~7000 K and the measurement of the ions by a mass spectrometer according to the mass-to-charge ratio (m/z).

Sample preparation

1. A sample of 0.2-0.5 g of dry matter into a Teflon vessel.
2. Microwave digestion in HNO₃ (6-8 ml) with H₂O₂ (1-2 ml) at a temperature of ~200 °C, pressure ~40 bar.
3. After cooling, making up with demineralised water to a reference volume (typically 25-50 ml).
4. Filtration or centrifugation before injection into the ICP-MS.

Internal standards

To correct for matrix effects, internal standards are added - typically ¹⁰³Rh, ¹¹⁵In, ²⁰⁹Bi at a concentration of a few to tens of μg/l.

Measured isotopes

As standard, ²⁰⁸Pb (lead), ¹¹¹Cd (cadmium), ²⁰²Hg (mercury), ⁷⁵As (arsenic) and ⁶⁰Ni (nickel) are monitored. If needed, others are added (⁵²Cr, ¹²¹Sb, ¹³⁷Ba).

Limit of quantification (LOQ) and its significance

LOQ is the lowest concentration of an analyte that a given analytical procedure can quantify with defined precision and accuracy (typically a relative standard deviation ≤ 10 %). For a method, the LOQ must be orders of magnitude below the applicable legislative limit - otherwise the method does not provide a reliable assessment.

Examples of typical LOQs:

• HPLC-DAD for mitragynine: 0.01 % by weight (of the order of 0.1 mg/g),
• HPLC-MS/MS for 7-OH-MG: a few ng/g of sample,
• ICP-MS for lead: 0.005 mg/kg (i.e. 0.005 μg/g) - three orders of magnitude below the legislative limit of 3 mg/kg.

Comparison with alternative methods

• GC-MS (gas chromatography) is less suitable for kratom alkaloids - the molecules are heat-labile and would require derivatisation. It is used for trace studies but is not the standard.
• UV-VIS spectrophotometry can provide the total alkaloid content but without separation of individual substances. It is not sufficient for the purposes of a COA.
• NMR allows structural identification, is used in research, but not for routine batch control.
• AAS (atomic absorption spectrometry) is an older method for heavy metals, now largely replaced by ICP-MS because of its lower sensitivity.

Frequently asked questions

Why not GC-MS?

Kratom alkaloids are heat-labile and would decompose in gas chromatography. HPLC is used for reliable determination.

What is the LOQ for mitragynine?

For HPLC-DAD, of the order of 0.01 % by weight; for HPLC-MS/MS, a few ng/g. The specific value depends on the particular method and instrument.

Can the content be measured at home?

Not reliably. Quantitative HPLC and ICP-MS require instruments costing hundreds of thousands to millions of crowns, calibration and metrological traceability. Indicative qualitative tests (e.g. colour reactions to alkaloids) have very limited evidential value.

Is a single method enough for the whole COA?

No. A comprehensive COA requires a combination of methods: HPLC for alkaloids, ICP-MS for metals, LC-MS/MS for mycotoxins, and cultivation methods for microbiology. An accredited laboratory has validated procedures for them.

References

• Wang M. et al. (2014). High-performance liquid chromatographic analysis of mitragynine and 7-hydroxymitragynine. Journal of Pharmaceutical and Biomedical Analysis, 90, 32-39.
• USP <233>. Elemental Impurities - Procedures. United States Pharmacopeia.
• ČSN EN ISO/IEC 17025:2018. General requirements for the competence of testing and calibration laboratories.