Hypromellose, or hydroxypropyl methylcellulose (HPMC), is a cellulose ether product. There are a large number of cellulose ethers with unique properties that are determined by the amounts and types of ether substitution. HPMC is a methyl and 2-hydroxypropyl ether of cellulose used in the pharmaceutical industry as an excipient and controlled-delivery component in oral medications and ophthalmic lubricant and an emulsifier and thickening agent in the food industry.
Because of its use in the pharmaceutical and food industries, the United States Pharmacopeia (USP) has established standards relating to the amount of methoxyl and 2-hydroxypropoxyl substitutions in HPMC. The USP implements a gas chromatography (GC) method for the determination of the ether content. Here we describe the validation of an assay using an Ultra Performance Liquid Chromatography (UPLC) method for the simultaneous analysis of the methoxy and 2-hydroxypropoxy content of HPMC that improves assay precision while decreasing inputs and cost.
Standard and sample preparations comply with those found in the current USP. The exception is that, unlike GC, UPLC technique is highly precise and does not require the addition of an internal standard for normalization.
To prepare HPMC for analysis, hydriodic acid is used to cleave the substituted methoxy and 2-hydroxypropoxy groups from the cellulose backbone and they are quantitatively converted into iodomethane and 2-iodopropane, respectively. This reaction is catalyzed by adipic acid and requires heating for one hour at 130˚C to complete the cleavage. The iodomethane and 2-iodopropane are then extracted by o-xylene. After cooling at 2-8˚C for 20 minutes to decrease pressure in the reaction vial and facilitate separation of the layers, the upper layer of o-xylene containing the iodomethane and 2-iodopropane is removed and resuspended in the appropriate amount of methanol for UPLC analysis.
For these studies we used HPMC sample substitution type 2910, USP grade. The standards were iodomethane (methyl iodide) ACS grade and iodopropane (isopropyl iodide) ACS grade. The standards were prepared as described above except they did not require heating.
We used the Waters Acquity H-class UPLC system with an Acquity UPLC BEH C18 1.7 µm, 50 x 2.1 mm column kept at 30˚C. Flow rate was 0.5 mL/min, injection volume 2 µL, and the UV detector at 254 nm. Samples were kept at 5˚C. Table 1 describes the UPLC gradient program used for these experiments.
The determination of methoxyl and 2-hydroxypropoxyl content in HPMC by UPLC was validated for specificity, precision, accuracy, intermediate precision, robustness, and solution stability. An example of Working Standard chromatogram is shown at Figure 1.
Instrument precision (system suitability) is shown in Table 2. The percent relative standard deviation (%RSD) of the working standard (instrument precision) was 0.2-0.6% for methyl iodide and 0.3-1.3% for isopropyl iodide peaks, respectively. Check Standard Agreement and %RD Bracketing Standard were within 0.1-1.1% for methoxyl iodide and within 0.1-1.3% for isopropyl iodide peaks, respectively.
No blank interference was observed across the elution windows of components (Figure 2).
An example of a HMPC sample run is shown in Figure 3, demonstrating the clear separation of the methyl and isopropyl iodide peaks in the sample.
The method precision and accuracy were evaluated on six individual HMPC sample preparations. The method precision was 0.9% for methoxyl and 1.2% for 2-hydroxypropoxyl components, respectively. The individual reportable assay results for each sample met USP specifications of 28-30% methoxyl and 7-12% 2-hydroxypropoxyl (Table 3).
Intermediate precision was performed on a different UPLC system by a second Analyst. The overall %RSD of the assay values from 12 HMPC replicates (Analyst 1 and Analyst 2) was 1.0% for the methoxyl component and 1.3% for the 2-hydroxypropoxyl component. Table 4 shows the % difference of the mean assay between Analysts 1 and 2 was 0.3% (absolute).
To test the robustness of the method we varied flow rate and column temperature in Robustness Tests 1-4 and found that the methoxyl and 2-hydroxypropoxyl results were still consistent with the original data (Table 5).
Standard and sample solutions were stable at room temperature for one day.
The UPLC method for the determination of methoxyl and 2-hydroxypropoxyl content of HPMC was successfully validated. The method was shown to be specific, precise, accurate and robust. The HPMC sample results were consistent with USP requirements for substitution type 2910. We also demonstrated that standard and sample solutions were stable for one day stored at room temperature.
UPLC technique results in increased resolution, speed and sensitivity. In contrast to the GC method, it requires no internal control. In addition, sample analysis only requires 6 minutes, allowing many samples to be run simultaneously. Each sample run consumes less mobile phase than traditional HPLC and requires low injection volume. This UPLC method allows for faster turnaround at comparable or lower costs.
Natalia Belikova, Willard Laster, Ervin Rozsypal, Gayla Velez
SGS Life Sciences
1. "Alternative Liquid Chromatographic Method for Determination of the Methoxyl and 2-Hydroxypropoxyl Content in Cellulose Ether Derivatives." J. Rashan, R. Chen, T. Zelensky and S. Sekulic. Journal of AOAC International, vol. 86, No. 4, pp. 694-702, 2003.
2. ICH Q2 (R1) Validation of Analytical Procedures, Text and Methodology
3. USP 38 Hypromellose Monograph