Determination of Mogroside V in Luohanguo Extract for Daily Quality Control Operation Using Relative Molar Sensitivity to Single-Reference Caffeine

Abstract

Mogroside V is one of the characteristic and effective components of luohanguo extract, a food additive used as a sweetener in Japan as per Japan’s Standards and Specifications for Food Additives (JSFA; 9th ed.). JSFA stipulates that the quantitative determination for mogroside V content in luohanguo extract applies HPLC using analytical standard mogroside V. However, no mogroside V reagents with proven purities are commercially available. Therefore the current JSFA determination method is not particularly suited for daily quality control operations involving luohanguo extract. In this study, we applied an alternative quantitative method using a single reference with relative molar sensitivity (RMS). It was possible to calculate the accurate RMS by an offline combination of ¹H-quantitative NMR spectroscopy (¹H-qNMR) and an HPLC/variable-wavelength detector (VWD). Using the RMS of mogroside V to a commercial certified reference material grade caffeine, the mogroside V contents in luohanguo extracts could be determined using HPLC/VWD without analytical standard mogroside V. There was no significant difference between the mogroside V contents in luohanguo extracts determined using the method employing single-reference caffeine with the RMS and using the JSFA method. The absolute calibration curve for the latter was prepared using an analytical standard mogroside V whose purity was determined by ¹H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative determination of mogroside V in luohanguo extract and can be used as an alternative method to the current assay method in JSFA.

Introduction

Many useful natural products, such as crude drugs and food additives, are standardized to control their efficacy and quality for safe use. Monographs for food additives including their definition, contents of constituents, identification tests, and standards for their use, are set in Japan’s Standards and Specifications for Food Additives (JSFA; 9th ed., 2018). Generally, HPLC with a variable-wavelength detector (HPLC/VWD) is employed as a quantitative method for natural products because of its high separation capability and sensitivity. Effecting HPLC/VWD requires an analytical standard that is identical to the analyte employed and that can be certified in terms of its exact purity. However, there are few commercially available certified reference materials (CRMs) with purities metrologically calculated by appropriate methods.¹⁾ In many cases, reagents conforming to the standards of manufacturers are used as analytical standards due to lack of CRMs. The purities of these reagents are guaranteed only by the standards employed by their manufacturers and are not necessarily metrologically accurate. This substitution gives rise to unreliable quantification. Furthermore, most of the constituents derived from natural products are not commercially available, including CRMs. In these cases, quantitative methods cannot be established for quality control, thereby preventing development of specification for food additives, particularly for those that are naturally derived.

Luohanguo extract, a sweetener obtained from the fruit of the luohanguo plant (Siraitia grosvenorii [Swingle] C. Jeffrey ex A.M. Lu and Zhi Y. Zhang [Cucurbitaceae]), is a food additive listed in JSFA.²⁾ In dried luohanguo extract, mogroside V(1) has to be present at a rate of more than 20%²⁾ (see Fig. 1). To obtain the required quality, quantitative assay of mogroside V by chromatography is required. Commercially available mogroside V reagents vary in price from ¥11000 to ¥110000 per 100 mg (as of February 2020), and their grade varies from reagent to analytical standard. When a reagent without exact purity is used as a standard for quantitative determination by HPLC, the purity of the reagent is generally regarded as 100%. Even if an identical sample is quantified, the resulting value without correction by purity of the standard may vary depending on the reagents used as standard. For quantitative determination according to JSFA, mogroside V standard must conform to the reagent specification in these guidelines. Conforming reagents are necessarily more expensive because reagent manufacturers are responsible for the certification of their reagents in accordance with JSFA specifications. Quality control of luohanguo extract is routinely carried out; therefore using expensive reagents for quantitative assay represents a heavy burden for manufacturers of food additives. Furthermore, even if reagents conforming to specifications are used, accurate quantification of mogroside V cannot be achieved because the specification does not consist of testing for determining the exact purity of the reagent. It is uncertain whether mogroside V standards that conform to specifications will be stably provided in the future. However, determination of mogroside V for quality control must be continued, as long as luhanguo extracts are used as sweeteners in Japan. This is a common problem concerning the constituents of natural products.

Fig. 1. Structures of Mogroside V and Caffeine

To overcome this problem, we developed a single-reference HPLC method with relative molar sensitivity (RMS) (Fig. 2 (A)). In chromatography, the detector’s response is generally proportional to the amount, or concentration, of analyte, and the response intensity per unit amount is substance specific. The RMS is defined as the ratio of response intensity per mole of an analyte to that of a reference compound (i.e., a stable and appropriate compound that is different from the analyte). As long as the RMS is applied to quantitative assays under the same HPLC conditions as those for RMS determination, it can be used with a specific factor. Hence using the RMS of an analyte to the reference, the analyte content in a sample solution (the constant amount of single reference added in advance) can be quantified using HPLC/VWD, based on the relationship between the ratio of detector response intensities, the amount of the single-reference, and the RMS.

Fig. 2. Principle of Single-Reference HPLC Method with Relative Molar Sensitivity (RMS)

(A) Single-reference HPLC method with RMS for determination of mogroside V in luohanguo extract. With the aid of the RMS of mogroside V to caffeine, we can obtain the exact mogroside V content (n_M) in luohanguo extract without the analytical standard of mogroside V by knowing the amount of reference compound in the external standard solution (n_c) and peak areas of mogroside V and caffeine (A_M and A_c) from HPLC chromatograms. (B) Schematic representation of RMS determination using off-line combination of ¹H-quantitative nuclear magnetic resonance spectroscopy (¹H-qNMR) and chromatography. a_M and a_c are slopes of calibration curves of mogroside V and caffeine. (Color figure can be accessed in the online version.)

An accurate determination of the RMS can be achieved by the offline combination of ¹H-quantitative NMR spectroscopy (¹H-qNMR) and HPLC/VWD (Fig. 2(B)). ¹H-qNMR is an International System of Units (SI) traceable measurement for the quantitative analysis of organic compounds^3,4); it is, however, inferior to chromatography in terms of sensitivity and multi-compound separations. Because the detection sensitivity of ¹H-qNMR is proportional only to the number of ¹H nuclei, analyte purity can be accurately estimated by comparing the signal areas of the analyte with those of a CRM that is prepared for ¹H-qNMR and used as an internal standard.^5,6) Since exact purities of reagents at any grade can be calculated by ¹H-qNMR, the accurate calibration curves of analytes can be constructed with HPLC/VWD. From the ratio of slopes of these accurate calibration curves of an analyte and a single-reference, an accurate RMS can be obtained. This offline combination of ¹H-qNMR and HPLC/VWD enables the single-reference method with RMS, which does not require analytical standards corresponding to the analytes for quantitative assays. As shown in our previous studies,^7–10) we applied the single-reference method with RMS to the quantitative determination of active constituents in various natural food additives.

In this study, we report that the single-reference HPLC method with RMS was applied for the quantitative determination of mogroside V in luohanguo extract using caffeine as a single reference. In the context of the official method, it is essential that anyone must be able to perform quantitative determination appropriately using any reagent and instrument, regardless of their manufacturers, as long as the reagent and instrument meet the specifications. In the single-reference HPLC method, accurate RMS determined by ¹H-qNMR is provided alongside the procedure, and a compound available as a commercial standard with certified purity is selected as a reference compound. This method is sufficiently accurate to serve as an alternative for the quantitative assay defined by JSFA.

Results and Discussion

Selection of Single Reference and Establishment of HPLC Conditions

At first, a single-reference compound was selected. It is essential for this reference compound to be chemically stable, to have certificated purity, and to be low cost. It is also preferable for the reference compound to have the same or close absorption maximum (λ_max) to the analyte for reducing the influence brought on by differences in HPLC instruments.⁹⁾ In this study, caffeine (2) was selected as a single-reference compound candidate to meet these requirements (Fig. 1); caffeine is available as a CRM grade reagent with certified purity. Moreover, the λ_max of caffeine is close to that of mogroside V, the analyte, although the respective λ_max do not exactly match each other.

Secondly, the HPLC conditions were determined. The JSFA prescribes HPLC analysis for the determination of mogroside V content in luohanguo extracts to be performed under isocratic elution condition with an amino column. This HPLC condition was changed into the appropriate isocratic conditions with caffeine as a single-reference compound. However, the retention time of caffeine was apart from that of mogroside V, leading to prolonged elution time for both compounds when they were analyzed under an HPLC isocratic elution condition. Considering this method is defined as a JSFA official method and used for daily operations, it is preferable for one analysis to be conducted within roughly 30 min. Therefore we propose a different HPLC condition for caffeine (which was used as an external standard) from that for mogroside V and luohanguo extract (Table 1).

Table 1. Operational Conditions for HPLC/VWD

Column	L-column2 ODS (5 µm, 4.6 × 250 mm) (CERI, Tokyo, Japan)
Column temperature	40 °C
Injection volume	10 µL
Mobile phase	A: water/formic acid (1000 : 1), B: acetonitrile/formic acid (1000 : 1)
	Mogroside V and luohanguo extract	A : B (78 : 22)
	Caffeine	A : B (90 : 10)
Flow rate	1.0 mL/min
Detector wavelength	210 nm

¹H-qNMR Measurement for Purity Assessment of Mogroside V Standard

The RMS was determined from the ratio of slopes in the calibration curves for the analyte and reference compound; here, horizontal and vertical axes in the calibration curves were molar concentration (µmol/mL) and peak area, respectively. To determine the accurate RMS value of mogroside V to caffeine, accurate calibration curves were needed. These could be constructed using reagents whose exact purities were available. The given purity of the caffeine reagent used as a single-reference compound in this study was reliable because of its availability as a CRM reagent, while that of mogroside V standard was unclear. To establish accurate calibration curves, purity of the mogroside V standard was determined by ¹H-qNMR using internal standard.^5,6)

The ¹H-qNMR spectrum of solution^NMR1 is shown in Fig. 3. In this spectrum, chemical shifts were defined as a ¹H signal derived from the methyl group of DSS-d₆ calibrant as δ0 ppm. In addition, ¹H signals derived from saturated hydrocarbons of aglycone moiety were detected at roughly δ 0.8 to 3.0 ppm, whereas ¹H signals derived from glycosides were observed at approximately δ 3.2 to 5.0 ppm (Fig. 3). However, these signals were inappropriate as quantitative signals used for ¹H-qNMR, due to overlapping with other ¹H signals. The signal at δ5.59 ppm from H-6 of mogroside V, however, was accepted as suitable as the quantitative signal for mogroside V because it showed good separation from other signals derived from the analyte, 2,2-dimethyl-2-silapentane-5-sulfonate-d₆ sodium salt (DSS-d₆) calibrant, and impurities (Fig. 3). Using this signal in the ¹H-qNMR spectra of solutions^NMR 1 to 3, the absolute purity of the mogroside V standard was determined as 93.8% (relative standard deviation (RSD) 0.2%).

Fig. 3. ¹H-Quantitative NMR Spectroscopy (¹H-qNMR) Analysis of Mogroside V Standard

The H-6 signal at δ5.59 ppm of mogroside V was used as a quantitative signal.

Determination of RMS of Mogroside V to Caffeine

To determine the accurate RMS value of mogroside V to caffeine, accurate calibration curves were constructed to correct the molar concentration on the horizontal axes with certified purity of caffeine (99.9%) or purity of mogroside V (93.8%), as determined by ¹H-qNMR (Fig. 4). The resultant RMS values determined by the ratios of the slopes of curves are shown in Table 2. The average RMS of mogroside V to caffeine among three chromatographs (chromatographs A to C) was 0.127, and RSD of the RMS values was 4.2%. The variation of RMS values obtained from different instruments was assumed to have occurred due to discrepancy of detector wavelength (at 210 nm) and the λ_max of mogroside V (under 200 nm). In many cases, the curve of the absorption spectra was relatively shallow near λ_max, reducing the influence of the difference in the absorption spectral resolution on the intensity of detector responses. However, for the official method, setting the detector wavelength below 200 nm was avoided; the reason for this was linked to perspectives of the influences caused by the absorption of mobile phase and differences in the sensitivity of detectors. Since differences in the RSD of RMS values due to difference of instruments were within a 5% margin in this study, a detector wavelength at 210 nm was considered appropriate for adopting the single-reference HPLC method as a quantitative assay in JSFA.

Fig. 4. Calibration Curves of Mogroside V and Caffeine with Three Chromatographs

The concentrations of standard solutions reflected the purities of mogroside V standard and caffeine (CRM).

Table 2. Relative Molar Sensitivities of Mogroside V to Caffeine Based on Three Chromatographs

Chromatograph		Calibration curve slope		RMS	RSD (%)
		Mogroside V	Caffeine
A	1	1832592	13623068	0.133	0.6
	2	1816983	13635516
	3	1812307	13715035
B	1	1561	12785	0.121	1.2
	2	1534	12686
	3	1539	12951
C	1	1624695	12698457	0.127	0.7
	2	1616316	12682011
	3	1600585	12615545
Average of RMSs for three chromatographs				0.127	4.2

In the determination of mogroside V content in luohanguo extracts using the current method described in JSFA (JSFA method), the purity of any commercial mogroside V standard is regarded as being 100%. Employing the mogroside V standard used in this study (purity 93.8%), a minimum 6% higher content of mogroside V compared with true content will be calculated using the JSFA method. Since the purity of mogroside V standards can vary significantly according to lot number and manufacturer, different figures can be obtained for mogroside V content from identical luohanguo extract products, undermining their consistent quality control. In contrast, the single-reference HPLC method developed in this study required caffeine as a single reference. The CRM grade of caffeine was available, and as a result, mogroside V content was rarely affected by differences linked to lot numbers and manufacturers where reagents were concerned. Although RMS differences based on instruments affected the content of mogroside V, these were within a 5% margin for the conditions in this study. These differences were smaller than those between the true amount of mogroside V and that calculated by the JSFA method using mogroside V standards. Moreover, the margin for RSD of RMSs obtained by the same instrument was within 1.2% (Table 2). These results indicate that the single-reference HPLC method developed in this study is suitable for daily operation in the quality control of luohanguo extract products, compared with the JSFA method.

Comparison of Single-Reference HPLC Method with JSFA Method

For determining mogroside V content, luohanguo extract products C2101 and C2102 were analyzed using the single-reference HPLC method. Quantitative determination with the single-reference HPLC method was performed using three instruments (chromatographs A to C). Figure 5 shows the chromatograms of mogroside V, caffeine, and two luohanguo extract products analyzed by chromatograph A. For a more exact quantitative analysis, the concentration of the caffeine standard solution used as an external standard was corrected using certified purity (99.9%) and the contents of mogroside V in luohanguo extract products were determined (see Table 3).

Fig. 5. HPLC Chromatograms of Mogroside V Standard Solution (A), Caffeine Solution (B), and Two Luohanguo Extract Products (C, D) Analyzed with Chromatograph A Using the Single-Reference HPLC Method

Peaks 1 and 2 are mogroside V and caffeine, respectively.

Table 3. Mogroside V Content of Luohanguo Extract Determined by Single-Reference HPLC Method, Absolute Calibration Curve Method, and JSFA Method

A. C2101
Chromatograph	A	B	C	Average	RSD (%)
Single-reference HPLC method	33.0%	30.4%	32.1%	31.8%	1.3%
Absolute calibration curve method	33.6%	34.2%	33.9%	33.9%	0.3%
Absolute calibration curve method (corrected by 1H-qNMR)	31.5%	32.1%	31.8%	31.8%	0.3%
JSFA method	33.6%	—	—	33.6%	—
JSFA method (corrected by 1H-qNMR)	31.1%	—	—	31.1%	—
B. C2102
Chromatograph	A	B	C	Average	RSD (%)
Single-reference HPLC method	59.8%	54.0%	56.7%	56.8%	5.1%
Absolute calibration curve method	61.0%	60.9%	60.0%	60.6%	0.9%
Absolute calibration curve method (corrected by 1H-qNMR)	57.2%	57.1%	56.3%	56.8%	0.9%
JSFA method	61.6%	—	—	61.6%	—
JSFA method (corrected by 1H-qNMR)	57.0%	—	—	57.0%	—

To verify the single-reference HPLC method, the mogroside V contents of C2101 and C2102 were also analyzed using not only the JSFA method (Fig. 6) but also a calibration curve of mogroside V instead of RMS, using the same conditions as the single-reference HPLC method (absolute calibration curve method). The resultant figures were compared with those using the single-reference HPLC method (Table 3).

Fig. 6. HPLC Chromatograms of Mogroside V Standard Solution (A) and Two Luohanguo Extract Products (B, C) Analyzed by Chromatograph A Using the JSFA Method.

Peak 1 is mogroside V.

The content of mogroside V in C2101 determined by the JSFA method and the absolute calibration curve method was 33.6 and 33.9%, respectively. In the case of C2102, the mogroside V content determined by these two methods was 61.6 and 60.6%, respectively. Although the HPLC elution conditions were different between the JSFA method and the absolute calibration curve method, the content of mogroside V was almost identical, indicating that the elution condition developed in this study was appropriate for quantitative determination. However, the mogroside V content obtained using these two methods was likely higher than the actual content because the purity of mogroside V standard used was regarded as 100%.

For a more exact quantitative analysis, the concentration of mogroside V standard solutions used as an external standard in the JSFA method and for the calibration curve in the absolute calibration curve method was corrected using the purity of the mogroside V standard determined by ¹H-qNMR. The content of mogroside V in C2101 determined by the modified JSFA method and modified absolute calibration curve method (both methods with correction by ¹H-qNMR purity) was 31.1 and 31.8%, respectively, whereas that in C2102 determined by these two modified methods was 57.0 and 56.8%, respectively (Table 3). From the average of these figures, the exact content of mogroside V in the two luohanguo extract products used in this study was accepted as 31.5% in C2101 and 56.9% in C2102 (we defined these values as “the exact content”).

The content of mogroside V in C2101 and C2102 as determined by the single-reference HPLC method was 31.8 and 56.8%, respectively. These results were almost identical to the exact content. When using the same instrument, the content determined by the single-reference HPLC method was found to be closer to the exact content than that determined by the absolute calibration curve method without correction by ¹H-qNMR purity. These results demonstrate the single-reference HPLC method developed in this study as a superior quantitative method to the JSFA approach and the absolute calibration curve method, in which the purity of the mogroside V standard was regarded as 100%.

The RSD of the content determined by the single-reference HPLC method using three chromatographs was 4 to 5%, which was larger than the RSD achieved by the absolute calibration curve method. Although the RMS determined in this study was 0.127, RMSs of chromatograph A and B were 0.133 and 0.121, respectively, and these differed by 0.006 from the RMS used in this study. Since the contents were obtained by multiplying the reciprocal of RMS (Eqs. (3) and (4)), the larger value was calculated from chromatograph A and the smaller value from chromatograph B. In contrast, for the absolute calibration curve method, calibration curves were constructed using the same mogroside V standard among three chromatographs. This means that the difference between chromatographs had been corrected and the difference in amounts derived from three chromatographs was narrowed. Practically, the lot numbers and manufacturers of mogroside V standards are not always the same; therefore the differences between instruments were thought to be larger than the results achieved in this study. In addition, the difference between the exact content and the measured value was 4.6% maximum in the single-reference HPLC method but 8.5% in the absolute calibration curve method (C2101). Although the influence of different instruments on quantification by the single-reference HPLC method will be one of the issues for future research, its influence on daily operations for quality control of luohanguo extract products appears to be small.

Using analytical standards with exact purities, accurate calibration curves can be constructed correctly to quantify mogroside V content. However, as long as standards with exact purities are not commercially available, analysts themselves will have to determine the exact purity of the standard using proper methods such as ¹H-qNMR. Since NMR instruments are less widely available than HPLC instruments, it would be difficult for some manufacturers to determine the exact purities of standards in daily operations for quality control of their products. Using the single-reference HPLC method developed in this study, more analysts can determine the exact content of mogroside V in luohanguo extract products using only HPLC instruments and without the exact purities of mogroside V standards. This method is suitable as a quantitative assay for luohanguo extract and can substitute for the current JSFA method.

Conclusion

This study aimed to introduce the single-reference HPLC method for determining the content of mogroside V in luohanguo extracts (without using mogroside V standards) as an official method in the JSFA. Caffeine, which is commercially available as a CRM, was selected as a reference compound. The RMS of mogroside V to caffeine was determined using an offline combination of ¹H-qNMR and HPLC/VWD. Using the RMS of 0.127, the quantitative determinations of mogroside V in two luohanguo extract products were performed with three chromatographs. More exact results were obtained using the developed method compared with the JSFA method. Data variation was within a 5% margin. A CRM grade caffeine reagent is available at ¥18800 per 100 mg, whereas a mogroside V standard without exact purity is ¥30000 per 50 mg (as of February 2020). The single-reference HPLC method with RMS using caffeine as an external standard was shown to be superior to the current JSFA method in terms of the accuracy of quantitative values and the cost of reagents. This method is thus suitable as a quantitative determination involving luohanguo extract and can be used as a substitute for the JSFA method.

Experimental

Materials and Reagents

Two commercial powdered luohanguo extracts, i.e., C2101 and C2102, used as food additives were obtained through the Japan Food Additive Association. According to product reports, the mogroside V content of these products was 32.57 (C2101) and 54.40% (C2102).

Mogroside V standard (Cat. no. 131-16571) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). CRM grade caffeine (Cat. no. 56396-100MG) was obtained from Merck KGaA (Darmstadt, Germany), and its certified purity was 99.9 ± 0.2%, k = 2 (Lot. no. BCBS0789V). In addition, 2,2-dimethyl-2-silapentane-5-sulfonate-d₆ sodium salt (DSS-d₆) (Cat. no. 044-31671, certified purity 92.3 ± 0.7%, k = 2 (Lot. no. KPE1040)), which was used as reference material for qNMR, was obtained from FUJIFILM Wako Pure Chemical Corporation Deuterium oxide (D₂O) (Cat. no. 151882-1KG) was purchased from Isotec (Miamisburg, OH, U.S.A.). Water was prepared using a water purification system (PURiC-ω; Organo Corp., Tokyo, Japan). All solvents used in this study were of HPLC or special grade.

Instruments

A 600 MHz NMR spectrometer (JNM-ECA600; JEOL Ltd., Tokyo, Japan) was used to determine the purity of mogroside V standard. Chromatographic separations were performed with three HPLC-VWD instruments (chromatographs A to C). Chromatograph A was a Prominence ultrafast LC system comprising a degasser (DGU-20 A_5R), a solvent delivery unit (pump, LC-20AT), an autosampler (SIL-20AC), a column oven (CTO-20AC), and an UV-visible detector (SPD-20 A) (Shimadzu Corp., Kyoto, Japan). Chromatograph B was an Agilent 1100/1200 Series comprising a degasser (G1322A), a binary pump (G1312A), an autosampler (G1329A), a column compartment (G1316A), and a VWD (G1314A) (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). Chromatograph C was a Dionex UltiMate 3000 comprising a degasser (SRD-3600), a dual-gradient standard pump (DGP-3600SD), an autosampler (WPS-3000TRS), a column compartment (TCC-3000SD), and a VWD (VWD-3400RS) (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). An ultramicrobalance (XP2U; Mettler Toledo International Inc., Columbus, OH, U.S.A.) was used for accurate measurement of weight.

Purity Measurement of Mogroside V by ¹H-qNMR

Mogroside V standard was dried at 105 °C for 2 h so as accurately to weigh out 40 mg into a vial. In the same vial, 1 mg DSS-d₆ was accurately weighed and dissolved in 4 mL of D₂O to obtain a sample solution for ¹H-qNMR (solution^NMR 1). This solution was assessed using ¹H-qNMR. The operating conditions of ¹H-qNMR were as follows: probe, Royal Probe (JEOL Ltd.); digital resolution, 0.25 Hz; spectral width, −5 to 15 ppm; spinning, off; pulse angle, 90°; ¹³C decoupling, on; relaxation delay, 60 s; number of scans 128; temperature, room temperature. Three sample solutions for ¹H-qNMR were prepared (solutions^NMR 1 to 3), and each sample solution was measured three times. As previously reported,^11–13) all ¹H-qNMR data were analyzed using Alice 2 for qNMR (JEOL Ltd.) and the purity of mogroside V (P_M)(w/w(%)) was calculated using the following Eq. (1):

  

(1)

where I is the ¹H signal area, H is the number of ¹H nuclei in one molecule contributing to I, M is molecular weight, W is the amount (mg) of reagent, and P is the certified value of purity (%). Subscripted M and DSS refer to mogroside V and DSS-d₆, respectively.

Determination of RMS of Mogroside V to Caffeine

Solution^NMR 1 was diluted with 70 vol% methanol to obtain mogroside V standard solutions for HPLC/VWD analyses at nine concentrations from 40 to 1300 µg/mL. In a volumetric flask, 22.1 mg of caffeine was accurately weighed and dissolved in water to create exactly 50 mL, followed by dilution to obtain caffeine standard solutions at nine concentrations from 1.1 to 36.4 µg/mL. These standard solutions were subjected to HPLC/VWD under the conditions shown in Table 1. A calibration curve of mogroside V was constructed using the concentrations corrected by purity, as determined by ¹H-qNMR, while that of caffeine was constructed with corrected concentrations by certified purity. Using the slope of the calibration curves (horizontal axis was corrected molar concentration (µmol/mL); the vertical axis was peak area), the RMS of mogroside V to caffeine was determined as follows:

  

(2)

where a is the slope of the calibration curve. Subscripted M and C refer to mogroside V and caffeine, respectively. In the same manner, RMSs were calculated using solutions^NMR 2 and 3.

Determination of Mogroside V Content in Luohanguo Extracts

Preparation of Sample and Standard Solutions

The luohanguo extract was dried at 105 °C for 2 h, and 0.2 g thereof was accurately weighed and suspended in 70 vol% methanol to create exactly 100 mL. The suspension was clarified through a membrane filter (0.45 µm pore size) to obtain the filtrate as a sample solution.

For preparation of the mogroside V standard solution, 5 mg of mogroside V standard (dried at 105 °C for 2 h) was accurately weighed and dissolved in 70 vol% methanol to make exactly 10 mL.

One of the caffeine standard solutions prepared in “Determination of RMS of Mogroside V to Caffeine,” the concentration of which was 18 µg/mL, was used as an external standard solution for quantitative determination.

Quantitative Determination of Mogroside V Content in Luohanguo Extracts Using RMS (Single-Reference HPLC Method)

Sample and standard solutions prepared in “Preparation of Sample and Standard Solutions” were subjected to HPLC under the conditions shown in Table 1. A peak of mogroside V in a sample solution was identified by comparing it with the peak observed in the mogroside V standard solution. Using the slope of the calibration curve (a_C) (horizontal axis was corrected caffeine concentration [µmol/mL]; vertical axis was peak area) and the peak area of mogroside V in the sample solution (A_M), the concentration (mg/mL) of mogroside V (C_M) in the sample solution was determined as follows:

  

(3)

where M is the molecular weight.

Using the concentration of mogroside V (C_M) obtained above, mogroside V content in luohanguo extract was calculated using the following Eq. (4):

  

(4)

where V is the volume (mL) of the sample solution and W is the amount (g) of luohanguo extract.

Quantitative Determination of Mogroside V by the Assay Defined in JSFA (JSFA Method)

Sample and mogroside V standard solutions prepared in ”Preparation of Sample and Standard Solutions” were subjected to HPLC according to the luohanguo extract assay listed in JSFA.²⁾ The HPLC condition is shown as follows: column, Shodex Asahipak NH2P-50 4E (5 µm, 4.6 × 250 mm) (Showa Denko K.K., Tokyo, Japan); column temperature, 40 °C; injector volume, 20 µL; mobile phase, acetonitrile/water (37 : 13); flow rate, 1.0 mL/min; and detector wavelength, 203 nm.

Using the peak areas (A_T and A_S) of mogroside V for the sample solution and mogroside V standard solution, the content of mogroside V was determined as follows:

  

(5)

where W_M is the amount (g) of mogroside V standard, W is the amount (g) of luohanguo extract, and P is the purity (%) of the mogroside V standard determined by ¹H-qNMR.

Acknowledgments

The authors are grateful for the assistance given by Mr. Go Kojima, Ms. Miyu Matsuda, and Ms. Akari Matsushita of the Department of Food Bioscience and Biotechnology at Nihon University. This work was partially supported by a Health and Labour Sciences Research Grant (No. H29-Syokuhinn-ippan-007) from Ministry of Health, Labour and Welfare, and Grant-in-Aid for Scientific Research (No. 16K18906) from the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

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