The Role of HPLC Testing in Verifying Botanical Purity

Abstract
The term "purity" is often misused in the botanical extract industry. High-performance liquid chromatography (HPLC), due to its high resolution, high sensitivity, and quantitative characteristics, has become the "gold standard" for verifying the purity of botanical raw materials under international dietary supplement, functional food, and cosmeceutical regulatory frameworks. This article uses Hericium erinaceus polysaccharides as a model to systematically explain the technical key points of HPLC in fingerprinting, polysaccharide homogeneity determination, adulteration screening, and stability testing, and demonstrates how Xi’an SOST Biotech Co., Ltd. integrates HPLC data into a closed-loop QA/QC system, thereby upgrading "purity" from marketing rhetoric to an auditable, traceable, and reproducible quantitative indicator, helping brands achieve smooth registration in North America, the EU, and the Asia-Pacific region without any supplementary submissions.

Industry Pain Points: Why is the "Purity" of Botanical Extracts Always Questioned?

Botanical raw materials are affected by differences in cultivation region, harvest season, drying methods, and extraction processes, resulting in batch-to-batch fluctuations of active ingredients exceeding 30%. Some suppliers specify "polysaccharides ≥30%", but use the UV method with glucose as an external standard, neither correcting for free sugar interference nor excluding starch dextrins, leading to actual measured immunologically active fragments (β-1,3/1,6-glucans) being less than 5%. When brands face GMP on-site inspections by the FDA/EFSA or COA random checks by Amazon/TikTok Shop, they are often deemed to have false labeling due to "methodological defects," leading to recalls.

Why is HPLC the Preferred Method over Traditional UV?

2.1 Separation Mechanism
HPLC, utilizing the distribution, adsorption, or size exclusion effects of the stationary and mobile phases, can baseline separate polysaccharides, oligosaccharides, monosaccharides, and excipients in a single chromatographic run, avoiding the false positives caused by the "one-size-fits-all" approach of the UV method.

2.2 Detection Limit and Quantification Limit
Taking pre-column derivatization HPLC-UV (PMP derivatization) as an example, the LOD of Hericium erinaceus characteristic oligosaccharides (DP3–DP8) can be as low as 0.02 mg L⁻¹, and the LOQ is 0.06 mg L⁻¹, which is far lower than the 1.5 mg L⁻¹ of the UV method.

2.3 Fingerprint Traceability
By establishing a "Reference Fingerprint," binding retention time, peak area ratio, and peak purity (PDA three-dimensional spectral matching degree ≥0.990), batch-to-batch consistency evaluation can be completed within 15 minutes, meeting the technical requirements of USP<2030> for "High-Resolution Chromatographic Fingerprint" of herbal medicines.

Example of Hericium erinaceus Polysaccharide HPLC Method Validation

3.1 Sample Preparation
The sample was prepared by hot water extraction at 80 ℃ → Sevag method deproteinization → 3 kDa ultrafiltration → vacuum concentration → freeze-drying, yielding a light yellow flocculent polysaccharide.

3.2 Chromatographic Conditions
Column: Waters XBridge Amide BEH (4.6×250 mm, 3.5 μm);
Mobile phase A: Acetonitrile, B: 15 mmol L⁻¹ ammonium acetate (pH 5.2); Gradient 75% A→55% A (0–30 min);
Flow rate: 1.0 mL min⁻¹; Column temperature 35 ℃; Injection volume 10 μL;
Detection: PDA 254 nm (after PMP derivatization).

3.3 System Suitability
Theoretical plate number ≥12000; Tailing factor ≤1.05; RSD (6 injections) ≤1.2%.

3.4 Linearity
An external standard curve was plotted using a mixed series of rhamnose-xylose-mannose-glucose-galactose (1:1:1:1:1) at different concentrations, with a correlation coefficient r≥0.9995.

3.5 Accuracy and Precision
Three-level spiked recovery rate: 98.7–101.4%, RSD ≤ 2.1%; intermediate precision (different dates, different analysts): RSD ≤ 2.6%.

3.6 Stability
The test solution was stored at 4 ℃ in the dark for 24 hours, and the peak area RSD ≤ 1.8%, meeting the requirements of ICH Q2(R1).

3.7 Fingerprint Spectrum
The similarity of 10 batches of raw materials (National Pharmacopoeia Commission "Traditional Chinese Medicine Fingerprint Spectrum Similarity Evaluation System" 2012 version) ≥ 0.98, confirming the stability of the process.

Four Major Application Scenarios of HPLC in Purity Verification

4.1 Polysaccharide Homogeneity Determination
Molecular weight distribution was determined by GPC-HPLC (TSKgel G5000PWXL), with a weight-average molecular weight Mw of 2.8 × 10⁵ Da and a polydispersity index Ð of 1.31, proving it to be a narrow-distribution homogeneous polysaccharide without starch fractions (Mw > 10⁷ Da) tailing.

4.2 Adulteration Screening
For the common adulteration with maltodextrin (DP≈7) in the market, a "peak area ratio" warning limit was established: DP7/DP3 ≤ 0.35.  Exceeding this limit triggers an OOS (Out of Specification) investigation.

4.3 Stability Study
After accelerated testing at 40 ℃/75% RH for 6 months, the HPLC fingerprint similarity was still ≥ 0.97, indicating that the characteristic oligosaccharides did not significantly degrade, supporting a 36-month shelf-life claim.

4.4 Regulatory Submission
The above data has been used by Xi’an SOST Biotech in the chemical characterization section of its New Dietary Ingredient Notification (NDIN) for Hericium erinaceus polysaccharide submitted to the US FDA, which passed the first round of FDA review without any questions.

Xi’an SOST Biotech: Making HPLC Reports a "Bidding Pass" for Customers

5.1 Equipment Investment
The company is equipped with 8 sets of Waters e2695 HPLC (including PDA, ELSD, and RI triple detectors) and 2 sets of Agilent 1290 UHPLC-QTOF high-resolution systems, with an annual testing throughput of >15,000 batches.

5.2 Quality System
Certified with ISO 9001, HACCP, USDA Organic, EU 2018/848 Organic, KOSHER, and HALAL; the QC laboratory complies with ISO/IEC 17025 guidelines and can provide complete COAs including original electronic data (Empower 3 audit trail).

5.3 Service Closed Loop
Customers can request "pre-shipment samples" before placing an order. The company provides a triple report including HPLC fingerprint, Mw distribution, and β-glucan content within 48 hours; after customer confirmation, the similarity between the bulk batch and the pre-shipment sample is ≥0.98, otherwise, a free rework is provided.

5.4 Customized Development
Targeting the "high immune activity" demand in the North American market, the company launched the "Hericium-MAX™" specification: β-1,3/1,6-glucan ≥25% (HPLC-ELSD quantitative analysis), which has successfully helped customers obtain the "#1 Best Seller" label on Amazon US.

5.5 Contact Information
Website: www.sostapi.com
Email: ericyang@xasost.com

Conclusion

HPLC is not only a testing method but also a "trust anchor" in the plant extract supply chain. Xi’an SOST Biotech deeply integrates HPLC fingerprinting, molecular weight distribution, and regulatory compliance, transforming "purity" into an auditable data package, giving customers a head start in market access, label claims, and consumer education. Choosing SOST means choosing data over stories and using standardization to mitigate risks.

References
[1] National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV): General Chapter 0512 High-Performance Liquid Chromatography.
[2] USP Convention. USP-NF 2024: <2030> High-Resolution Chromatographic Fingerprint Analysis for Botanicals.
[3] ICH Expert Working Group. ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology, 2005.
[4] Zhang A, et al. Structural elucidation and immunomodulatory activity of a β-glucan from Hericium erinaceus. Carbohydrate Polymers, 2022, 291: 119573.
[5] FDA. Guidance for Industry: New Dietary Ingredient Notifications and Related Issues (2022 Final Version).
[6] Xu W, et al. Study on the correlation between HPLC fingerprint and immunomodulatory activity of Hericium erinaceus polysaccharides. Chinese Traditional and Herbal Drugs, 2021, 52(15): 4452-4459.