Modernizing USP Melatonin Monograph Assay and Impurities Methods for Increased Throughput and Reduced Solvent Waste

Melatonin is a neurohormone that can regulate the sleep cycle in humans. It is readily available as dietary supplements and is often used for alleviating sleep-related disorders. Recent data has shown a dramatic increase in the number of incidents of pediatric Melatonin ingestion. Melatonin contents significantly higher than their labeled values in dietary supplement products have also been reported. To assist quality control of the Melatonin product manufacturing process, a modernized version of a USP Melatonin Monograph procedure has been developed on a Waters Arc^™ HPLC System using an XBridge BEH^™ C₁₈ Column (2.5 µm, 4.6 mm x 75 mm). The liquid chromatographic conditions were appropriately adjusted within the USP <621> guidelines, and the resulting separation performance meets the USP system suitability requirements, with the benefit of shorter run times and reduced solvent waste. 

Benefits

• Assay of Melatonin in a five minute isocratic run
• Quantification of impurities in a 13-minute gradient elution program
• Fulfillment of all system suitability requirements

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that is produced by the mammalian pineal gland. Since Melatonin plays an important role in the sleep cycle in vertebrates, it is often used for alleviating sleep-related disorders such as insomnia, anxiety, and jet lag.¹ Melatonin is readily available for purchase over the counter as dietary supplements in a variety of forms, including flavored liquid, chewable tablets, softgels, capsules, and gummies. Recently, it has been reported that the number of incidents of pediatric Melatonin ingestion increased 530% from 2012 to 2021 and these incidents were associated with 287 intensive care unit admissions and two deaths.² Recent studies also revealed that Melatonin content in some dietary supplements was found much higher than the label claim values. As high as 478% and 347% of the label value were found in a dietary supplement and a Melatonin gummy, respectively.^3,4 This has led to a real concern about the quality and safety of Melatonin-containing dietary supplements. Improved quality control is required for the dietary supplement manufacturers to ensure accurate label claims. The US Pharmacopoeia (USP) Melatonin Monograph provides industrial standards for the Melatonin assay and impurities.⁵ The goal of this application note is to modernize the USP methods on a Waters Arc HPLC System with a Waters 2998 PDA Detector using an XBridge BEH C₁₈  (2.5 µm, 4.6 mm x 75 mm) Column.

The standard and sample solution preparation and the Liquid Chromatographic (LC) conditions recommended by US Pharmacopoeia were adopted with minor adjustments.⁵ USP Melatonin RS, 5-Methoxytryptamine (5-MT) were purchased from Sigma-Aldrich (Allentown, PA).

Buffer: 0.5 g/L of monobasic potassium phosphate in water. Adjusted with phosphoric acid to a pH of 3.5 and filtered.

Standard solution: 0.1 mg/mL of USP Melatonin RS in mobile phase (acetonitrile and buffer 22/78 v/v).

System suitability solution: 0.1 mg/mL of USP Melatonin RS and 0.02 mg/mL 5-MT (Melatonin related compound A) in mobile phase (acetonitrile and buffer 22/78 v/v).

Sample solution: 0.1 mg/mL of Melatonin in mobile phase (acetonitrile and buffer 22/78 v/v).

USP Method Modernization

The USP Melatonin Monograph specifies a procedure for the assay and impurities of Melatonin. These methods were implemented on an Arc HPLC System with minor adjustments that were made under the USP allowable adjustment guidelines.⁶ A 2.5 μm particle size XBridge BEH C₁₈ Column was used for its higher separation efficiency per unit length, instead of a 5 μm particle column as in the USP Monograph. The column length was adjusted to 75 mm to keep the L/dp (column length to particle size ratio) the same as that in the USP monograph. The mobile phase of acetonitrile and buffer at 25:75 (v/v) was recommended by the USP Monograph, however, a small adjustment of the mobile phase composition to 22:78 (acetonitrile and buffer) was found necessary to satisfy the system suitability requirement on RRT (relative retention time) of the related compound A (5-MT) and Melatonin of 0.4 and 1, respectively. This small adjustment in composition was within the limit for adjustments under the USP guideline (less or equal than 10% absolute, or within 30% relative for the minor component of the mobile phase). The scaled flow rate for the 2.5 μm particle column was 2.0 mL/min based on the USP recommended flow rate of 1.0 mL/min for a 5 μm column (with the same column I.D.). However, based on our investigation of the separation performance (see Table 2), a flow rate of 1.0 mL/min was selected for the assay because of its optimal separation efficiency (plate count number) in the 1.0–2.0 mL/min range. Please note that the pre-heating tubing inside the column heater was by-passed when connecting the column to the injector. This helped with the separation efficiency by reducing the extra-column band spreading for this 75 mm length column (4.6 mm x 75 mm). Figure 1 and 2 show chromatograms of the Melatonin and the related compound A (5-MT) under the isocratic elution conditions (for assay) and the gradient elution conditions (for impurities). The run time was five minutes in the isocratic elution and 13 minutes in the gradient elution, respectively. These run times were about half of the run times in the USP methods. These short run times and the associated reduced solvent wastes would shorten the analysis turnaround time and reduce the operating cost. 

System Suitability Performance

The USP Melatonin Monograph specifies system suitability requirements for the assay and the related compounds. These requirements include the RRT of the related compound A and Melatonin, resolution between the related compound A and Melatonin, and relative standard deviation (RSD) on melatonin (see Table 3). Table 4 shows the system suitability performance and repeatability results for both Melatonin and its related compound A (5-MT) on the Arc HPLC System with an XBridge BEH C₁₈ Column (2.5 µm, 4.6 mm x 75 mm). RSD of 0.11% was obtained for the Melatonin peak area. The RRT values, the resolution, and the repeatability all met the USP system suitability requirements (See Table 3). 

This application note demonstrates a modernization of the USP Melatonin Monograph procedure using a Waters Arc HPLC System and an XBridge BEH C₁₈ Column (130 Å, 2.5 µm, 4.6 mm x 75 mm). The LC conditions were appropriately adjusted under the USP guidelines. The separation of Melatonin and the related compound A met all USP system suitability requirements, including the relative retention times, resolution, and relative standard deviation. The use of a 2.5 µm particle XBridge BEH C₁₈ Column (4.6 mm x 75 mm) offers short run time and reduced solvent consumption, which shortens the turnaround time and reduces operating costs.

1. Auld F, Maschauer EL, Morrison I, Skene DJ, Riha RL. Evidence for the Efficacy of Melatonin in the Treatment of Primary Adult Sleep Disorders. Sleep Med Rev. 2017;34:10–22. doi: 10.1016/j.smrv.2016.06.005. 
2. Lelak K, Vohra V, Neuman MI, Toce MS, Sethuraman U. Pediatric Melatonin Ingestions—United States, 2012-2021. MMWR Morb Mortal Wkly Rep. 2022;71(22):725–729. doi:10.15585/mmwr.mm7122a1.
3. Lauren A.E. Erland, Praveen K. Saxena. Melatonin Natural Health Products and Supplements: Presence of Serotonin and Significant Variability of Melatonin Content.J Clin Sleep Med. 2017 13(2):275–281. doi: 10.5664/jcsm.6462.
4. Cohen P.A., Avula B., Wang Y-H, Katragunta K., Khan I. Quantity of Melatonin and CBD in Melatonin Gummies Sold in the US. JAMA. 2023; 329(16): 1401–1402. doi:10.1001/jama.2023.2296.
5. Melatonin Monograph. United States Pharmacopeia, Docid: GUID-454646BE-F1DF-458C-9011-1FBBCCEFE5BC_4_en-US, 2023.
6. Chapter 621 Chromatography. United States Pharmacopeia, Docid: GUID-6C3DF8B8-D12E-4253-A0E7-6855670CDB7B_7_en-US, 2023.