Hptlc–densitometry Method For Simultaneous Estimation Of Boric Acid

HPTLC–densitometry method for simultaneous estimation of boric acid/borate and calcium fructoborate complex in the bulk, tablets and capsules dietary supplements

Andrei Bițăa, George Dan Mogoșanua, *, Ludovic Everard Bejenarua, Cornelia Bejenarub, Octavian Croitoruc, Gabriela Răud, Johny Neamțue, Iulia Daria Scoreif, Ion Romulus Scoreif, John Hunterg, Brad Eversg, Boris Nemzerg

aDepartment of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareș Street, 200349 Craiova, Romania

bDepartment of Vegetal & Animal Biology, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareș Street, 200349 Craiova, Romania

cDepartment of Drug Control, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareș Street, 200349 Craiova, Romania

dDepartment of Organic Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareș Street, 200349 Craiova, Romania

eDepartment of Physics, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova,
2 Petru Rareș Street, 200349 Craiova, Romania

fBioBoron Research Institute, University of Craiova, Mircești Street, Bldg. M4/1/1, 200506 Craiova, Romania

gVDF FutureCeuticals, 2692 N. State Rt. 1–17, Momence, 60954 IL, USA

*Corresponding author: Phone/Fax +40251–523 929, E-mail: [anonimizat]

E-mail addresses: [anonimizat] (A. Biță), [anonimizat] (G.D. Mogoșanu), [anonimizat] (L.E. Bejenaru), [anonimizat] (C. Bejenaru), [anonimizat] (O. Croitoru), [anonimizat] (G. Rău), [anonimizat] (J. Neamțu), [anonimizat] (I.D. Scorei), [anonimizat] (I.R. Scorei), [anonimizat] (J. Hunter), [anonimizat] (B. Evers), [anonimizat] (B. Nemzer).

Abstract

The paper describes a new, simple, precise, and accurate HPTLC–densitometry method for simultaneous estimation of boric acid (BA)/borate and calcium fructoborate complex (CFC) in the bulk dietary supplements and tablet/capsule dosage forms. Chromatographic separation was performed on silica gel 60 F254 pre-coated glass plates (stationary phase) and the solvent system consisted of 2-propanol–water 8:2 (v/v). Densitometry evaluation was performed at 365 nm. The two boron-based dietary supplements were satisfactorily resolved with Rf values 0.58±0.01 (CFC) and 0.71±0.01 (BA/borate). The accuracy and reliability of the method was assessed by evaluation of linearity (100–700 ng/band for BA/borate and 0.5–3.5 μg/band for CFC), precision [for BA/borate – intraday Residual Standard Deviation (RSD) 1.57–1.80, interday RSD 1.38–1.65; for CFC – intraday RSD 1.16–1.55, interday RSD 0.79–1.67], accuracy (98.5–101.5 for BA/borate and 99.2–99.6 for CFC), and specificity in accordance with International Conference on Harmonization (ICH) Guidelines.

Keywords: Boric acid; Borax; Calcium fructoborate complex; Dietary supplements; HPTLC.

1. Introduction

Boron exists in plant tissue as borate monoesters and diesters together with boric acid [1–3]. The formation of borate esters has been expected to be a key to the physiological function of boron [4–6]. At present, in the nutraceuticals industry, the available nutritional products are calcium fructoborate, boron citrate, boron aspartate, boron glycinate chelates, and boron ascorbate or boric acid and sodium borate [7]. Out of the boron-based nutraceuticals, the most studied from the scientific point of view and the only nutritional supplement that is found in nature, is calcium fructoborate, a natural sugar-borate ester (SBE) [8]. Among the compounds that are known to form stable borate esters, the borate formed by the complexation of boric acid (BA) with fructose and calcium, calcium fructoborate complex (CFC), has been shown to offer distinct potential for human health [9–11]. CFC is a patented plant mineral complex that is marketed as a nutritional supplement FruiteX-B® (FrxB) [12], nature-identical analogue of a plant mineral complex commonly found in fruits, vegetables, herbs, and wine with potential health benefits for conditions linked to inflammation such as bone, joint and cardiovascular system [10,11].

Quantification of boronic/boric/borate esters has been performed in the past using, liquid and solid 11B-NMR [13–15], TLC methods [8,16–19], HPLC coupled with UV or fluorometric detection [20], thermal analysis [20,21], XRD (X-ray Diffraction), FTIR, Raman and ICP (Inductively Coupled Plasma) spectroscopy [22] either alone or in combination with other compounds.

To our knowledge, no articles related to the HPTLC determination of BA/borate and CFC in dietary supplements dosage forms have been reported in literature. Only a quantitative 11B-NMR method has been developed to enable quantification of the amount of CFC present in dietary supplements tableted or encapsulated in combination with other dietary supplements [13,23]. Solid-state 13C-NMR and 11B-NMR spectra on commercial FrxB demonstrate that any free BA or borate that is present in the powder sample is intimately integrated into the mixture and no crystalline domains of these species exist and the broad signals expected from crystalline borate/BA are not observed. However, the liquid-state 11B-NMR has identified the three basic types of boron-containing molecule in aqueous solutions of commercial FrxB (resulted by water hydrolysis): free BA/borate, monoester borate and diester borate. The relative molar concentrations of these three types of boron-containing molecule have been found to be approximately 5%, 10% and 85%, respectively [13,23]. Moreover, thermal analysis of FrxB showed comes therefore as the calcium fructoborate found in natural products, as unique entity, calcium fructoborate diester (Fig. 1). Molecular formula of calcium fructoborate diester has been identified as Ca[(C6H10O6)2B]2·• 4H2O [22].

Fig. 1

This CFC (a bis-fructose ester of boric acid) has been detected in plants, fruits, seeds, honey, and some foodstuff and it is a naturally occurring part of the human diet [7]. Because CFC has been shown to be an efficient, nontoxic precursor of the borate anion, having multiple published studies on its numerous potential contributions to human health, nutritional supplementation with CFC offers significant benefits in support of healthy bone and joints, as well as for cardiovascular health [7,11,24].

In this paper, we report a new, simple, fast, specific, and precise HPTLC–densitometry method for the simultaneous determination of CFC and BA/borate in dietary supplement products, in bulk, tablets or capsules and free from interferences present in formulations. HPTLC is superior to other analytical techniques in terms of total cost and time for analysis. HPTLC is being incorporated at a high rate in pharmacopeias and Good Manufacturing Practices (GMPs) for botanical and synthesis of dietary supplements, and it is predicted that the use of HPTLC methods will also increase in worldwide pharmacopeias on dietary supplements [25–27].

2. Experimental

2.1. Chemicals and reagents

CFC standard was synthesized according to Hunter’s US Patent [12] and was provided by VDF FutureCeuticals (Momence, IL, USA). The purity of CFC was provided by multinuclear magnetic resonance (mNMR) using liquid and solid state 11B- and 13C-NMR [13]. Four different boron-dietary supplement products (A–D) with commercial FrxB were purchased from local diet food stores. Three raw materials, borax decahydrate, boric acid (Optibor®) and FruiteX-B® were purchased from their distributors with the exception for FruiteX-B®, which has been received from VDF FutureCeuticals. Product A (serving size one tablet) containing 108 mg CFC plus glucosamine sulfate and chondroitin sulfate. Product B capsules containing 225 mg CFC per capsule plus vitamin D. Product C (also in capsules) containing 108 mg CFC plus calcium, magnesium, zinc, manganese and silicon. Product D has small quantity of CFC (54 mg) plus glucosamine sulfate, methylsulfonylmethane, black tea and resin extracts. Boric acid for standards was procured commercially from Merck Millipore (Darmstadt, Germany). Chromatographic grade solvents and reagents, such as 2-propanol and water, were obtained from Merck, and chlorogenic acid from Sigma-Aldrich (Munich, Germany).

2.2. Preparation of standard and sample solutions

A stock solution of boric acid (# 1.00165.0100, Merck; CAS Registry # 10043-35-3; 99.5–100.5% purity) was prepared at a concentration of 1 mg/mL in water. CFC (VDF Future
Ceuticals; 2.7% Boron purity) stock solution was prepared at a concentration of 1 mg/mL in water, too. The standard solution for boric acid was obtained by diluting 2.5 mL of stock solution with 25 mL solvent (water) in order to achieve a 100 ng/μL concentration and the standard solution for calcium fructoborate complex was prepared by diluting 12.5 mL of stock solution with 25 mL solvent to achieve a 500 ng/μL concentration. Each of the products used had CFC or BA/borate in different labeled forms. Table 1 shows the exact labeling and amounts of boron active substance (mg/tablet or mg/capsule) of each analyzed product.

Table 1

A stock solution of each capsule sample was prepared by solubilizing the content after accurately weighing it in 50 mL of solvent (water). Before applying it on the plate, the stock solution was diluted by taking 1 mL of it and adding 1 mL of solvent. The FruiteX-B® sample was prepared by solubilizing 100 mg in 50 mL water. The boric acid (Optibor®) and borax samples were prepared by solubilizing 20 mg in 50 mL water.

2.3. Equipments

CAMAG TLC Scanner 3 (CAMAG, Muttenz, Switzerland) equipped with computer system and winCATS software (WinCATS 1.4.3) were used for quantitative evaluation. CAMAG UV cabinet was used for visualization. The images were acquired with Nikon Coolpix S8000 digital camera of 14.2 million effective pixels resolution, 10× optical zoom. TKA Lab Tower system was used for pure water – EDI 15 model, at 230C the resistivity is 18.2 MΩ×cm (Thermo Electron LED GmbH, Niederelbert, Germany). BANDELIN SONOREX SUPER heated ultrasonic bath (BANDELIN Electronic GmbH & Co. KG, Berlin) and EPPENDORF 5804 centrifuge (EPPENDORF, Hamburg, Germany) were also used for sample preparation.

2.4. HPTLC analysis

Analyses were performed on HPTLC silica gel G 60 F254 glass plates (20×10 cm, 0.2 mm layer thickness, Merck DC). The plates were pre-cleaned by development to the top with chloroform–methanol (1:1, v/v) and then activated for 30 min., at 1050C, prior to sample application. Standard and sample solutions were applied to the plate using semiautomatic TLC sample applicator CAMAG Linomat V, spray-on band applicator equipped with a 100 μL syringe and operated with 8 mm band length, application rate 50 nL/s, distance between bands 11 mm, distance from the side edge 20 mm, and distance from the bottom 10 mm. The volumes applied for BA and CFC standards were 1, 2, 3, 4, 5, 6 and 7 μL and 1 μL for samples.

The plates were developed to 30 mm beyond the origin with a mixture of 2-propanol–water 8:2 (v/v) (10 mL) as mobile phase in a vapor-equilibrated chromatographic tank (CAMAG 20×10 twin trough chamber) at room temperature (23±0.50C) before use. The development time was approx. 30 min., the temperature 23±0.50C and migration distance 40 mm (from the bottom edge). After development, the mobile phase was evaporated from the plate by drying with a hair dryer for three minutes. The plate was then sprayed heavily and evenly with 0.1% chlorogenic acid ethanolic solution and dried with a hair dryer for approx. 1 min. and then visualized at 365 nm [18]. The quantification of the standards and samples were performed by means of a CAMAG TLC Scanner III controlled by WinCATS 1.4.3 version software and the densitometer produced a calibration curve by linear regression of the weights and areas of the standard zone scans and automatically interpolated the weights of the sample zones from their scan areas. Linear scanning at 365 nm using a CAMAG TLC Scanner III with tungsten, deuterium and mercury vapor source, slit length 6 mm, slit width 0.9 mm, and scanning rate 10 mm/s was used to measure the zones of standards and samples.

Several parameters were evaluated, including mobile phase composition and stationary phase type (silica gel, cellulose, alumina, and polyamide layers) to obtain optimal thin layer efficiency. Commercially available precoated silica gel plates were chosen as the stationary phase. The chromatographic conditions were established after a number of preliminary experiments involving selection of the proper mobile phase and detection reagent. Different mobile phases were tested. The mobile phase resolved the BA/borate and CFC very efficiently and is shown in Fig. 2. The Rf value for BA/borate was 0.71±0.01 and for the CFC was 0.58±0.01. It was selected for detection of BA/borate and CFC, the spraying with 0.1% chlorogenic acid ethanolic solution. The method was used to determine and differentiate BA/
borate of CFC content in different dietary supplement products (bulk, tablets and capsules).

After development and drying of the plates, evaluation of both boron compounds was performed by scanning densitometry (365 nm) by means of a CAMAG TLC Scanner III controlled by WinCATS 1.4.3 software. Peak areas were recorded for all the peaks. The amount of BA and CFC was computed from the peak area by use of WinCATS 1.4.3 software (CAMAG).

Fig. 2

2.5. Validation of HPTLC–densitometry method

Validation of the optimized HPTLC method was performed in terms of the following parameters: specificity, linearity and range, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ), and robustness.

2.5.1. Specificity

The specificity of the method was been ascertained by analyzing standard and sample of BA/borate and CFC. The bands for BA/borate and CFC in the samples were confirmed by comparing the Rf and spectra of the band with that of standards. The peaks purity of BA and CFC were assessed by comparing the spectra at three different levels; i.e., peak start (S), peak apex (M) and peak end (E) positions of the spot. For purity test, UV–azomethine H method [28] and 11B-NMR spectra [13] have also been achieved confirming the presence of BA/borate and CFC. Analysis of the unspiked blank solution showed that no interference occurred at the Rfs of CFB and BA/borate, and therefore, no correction of the scan areas of the spiked blank solution was required.

2.5.2. Linearity and range

The linearity of the method for BA/borate and CFC was checked between 100 and 700 ng/band, respectively 500 and 3500 ng/band and concentration was plotted against peak area. Aliquots of 1, 2, 3, 4, 5, 6, 7 μL of each working standard solution were spotted in triplicate on a HPTLC plate to obtain a final concentration of 100–700 ng per band, respectively 500 and 3500 ng/band and developed in conditions established in the method optimization, i.e., plate type and mobile phase [2-propanol–water 8:2 (v/v)].

The calibration curves were constructed by plotting the peak area versus the corresponding concentration of BA and CFC.

2.5.3. Precision

Precision was assessed by determination of repeatability and intermediate precision and was expressed in terms of Residual Standard Deviation (RSD). The low RSD values indicated the suitability of this method for routine analysis of BA and CFC in pharmaceutical dosage forms. Repeatability was performed by sample application and measurement of peak areas of six replicates of same concentration 200 ng/spot of BA and 1000 ng/spot CFC. It was expressed as RSD and standard error of the peak areas. The intra- and inter-day precision for evaluation of the method variability were determined from the concentration of standard solutions (200, 400, and 600 ng/band for BA and 1000, 2000, 4000 ng/band for CFC) for three times repeated within the same day and on three consecutive days, respectively. The results were expressed as RSD.

2.5.4. Accuracy

Accuracy, as recovery, was determined by the standard addition method. Pre-analyzed samples of boric acid (200 ng/band) and calcium fructoborate complex (1000 ng/band) were spiked with extra BA and CFC standard (0, 50, 100, and 150%) and the mixtures were reanalyzed. Percentage recovery and RSD were calculated for each concentration level.

2.5.5. LOD and LOQ

Limit of detection (LOD) and limit of quantification (LOQ) were determined by Standard Deviation (SD) method. They were determined from the slope of the calibration (S) curve and SD of the blank sample using following equations:

LOD = 3.3 × SD/S

LOQ = 10 × SD/S

were defined as the amounts for which signal to noise (S/N) ratios were 3 and 10, respectively.

2.5.6. Ruggedness and robustness

Ruggedness is a measure of the reproducibility of a test result under normal, expected operating conditions from instrument to instrument and from analyst to analyst. Ruggedness is regarded as an inter-laboratory property. Ruggedness of the proposed HPTLC method was determined in two different laboratories, with different analysts, different instruments, different batches of reagents, different elapsed assay times, different assay temperatures. These are the Laboratory of Pharmacognosy & Phytotherapy of the University of Medicine and Pharmacy of Craiova (Romania) and the Laboratory of the VDF FutureCeuticals (USA).

Robustness is a measure of the capacity of a method to remain unaffected by small but deliberate variations in method conditions, and is an indication of the reliability of the method. Robustness, as defined herein, is an intra-laboratory property. Robustness of the proposed HPTLC–densitometry method was determined to evaluate the influence of small deliberate changes in the chromatographic conditions during determination of BA and CFC (mobile phase composition, development distance, saturation time). The robustness of the method was evaluated by observing the influence of small variations in the composition of mobile phase 2-propanol–water (the chromatographic parameters were interchanged within the range of 1–10% of the optimum recommended conditions). The time from spotting to chromatography and from chromatography to scanning was varied from ±5 min. The robustness of the method was determined at a concentration level of 200 ng/band for BA, respectively 1000 ng/band for CFC by applying on plate in triplicate. The effect of these variations in experimental conditions on the peak areas was evaluated by calculating the RSD for each parameter.

3. Results and discussion

3.1. Optimization of HPTLC–densitometry method

The chromatographic conditions were established after a number of preliminary experiments involving selection of the proper mobile phase and detection reagent. Different mobile phases were tested. The mobile phase composition was optimized to establish a suitable and accurate HPTLC–densitometry method for BA and CFC analysis. The mobile phase 2-propanol–water 8:2 (v/v) resulted in sharp, symmetrical, and well-resolved peaks at Rf values of 0.58 and 0.71 for CFC and BA/borax, respectively (Fig. 2). For detection was selected 0.1% chlorogenic acid in ethanol. UV spectra measured for the chromatographic bands showed maximum absorbance at approximately 365 nm. Boric acid and borax (BX) have the same Rf and the same calibration curve (Table 2). The method was used to determine BA/BX and CFC content in different dietary supplement products (bulk, tablets and capsules) (Table 1).

3.2. Calibration plots

The calibration plots of peak areas against amount of BA/BX and CFC were linear in the range of 100–700 ng/band and 500–3500 ng/band, respectively. Linear regression data for the plots confirmed the good linear relationship (Table 2). The correlation coefficients (R2) 0.9949 and 0.9945, respectively, were highly significant (p<0.05). The linear regression equations were y=306.62 + 16.85x and y=480.85 + 1981.54x, respectively, where y is the response and x is the amount of BA and CFC.

Table 2

3.3. Quantitative determination of boron in dietary supplements by HPTLC–densitometry

1 μL aliquots of the treated samples were applied to the HPTLC plate as bands, along with standard solutions of BA and CFC. The identity of BA and CFC in water samples was confirmed by comparing the retention values (Rf) and UV spectra of the reference peaks with the corresponding peaks from the samples. The corresponding peaks were determined on the Rf values of 0.71±0.01 for BA and 0.58±0.01 for CFC, respectively (Table 3). The HPTLC densitogram of a standard mixture with the corresponding Rf values is presented in Fig. 2, illustrating the separation of the BA and CFC was been measured at 365 nm. Experimental results of BA and CFC amounts in tablets and capsules, expressed as a percentage of label claims, were in good agreement with the label claims, thereby suggesting that there is no interference from any of the excipients normally present in tablets/capsules. Seven different batches of boron-based dietary supplements were analyzed using the proposed procedures (Table 3, Fig. 3).

Table 3

Fig. 3

3.4. Validation of the method

3.4.1. Specificity

The peak purity of BA and CFC was assessed by comparing their respective spectra at the peak start, apex, and peak end positions of the band, that is r(S, M)=0.9971 and r(M, E)=
0.9973. A good correlation (r=0.9989) was also obtained between the standard and sample spectra of BA and CFC, respectively.

3.4.2. Linearity

The response was linear (R2 of 0.9949 for BA and 0.9945 for CFC) over the concentration range between 100–700 ng/band for BA and 500–3500 ng/band CFC, respectively (Table 2).

3.4.3. Precision

Results from determination of repeatability and intermediate precision, expressed as RSD are shown in Table 4. The developed method was found to be precise as the RSD values for repeatability and intermediate precision studies were <2%, as recommended by International Conference on Harmonization (ICH) Guidelines [29].

Table 4

RSD for BA was in the range 1.57–1.80 for repeatability and 1.12–1.65 for intermediate precision, respectively for CFC was in the range 1.16–1.55 for repeatability and 0.79–1.67 for intermediate precision. These low values indicate the method is precise [22].

3.4.4. Accuracy/recovery studies

As shown from the data in Table 5, good recoveries of BA in the range from 92 to 98.5% and CFC in the range from 88.08 to 99.4% were obtained at various added concentrations with RSD values in the range 1.26–1.77 for BA and 1.6–1.89 for CFC. These results indicated that the method was accurate (Table 5).

Table 5

3.4.5. LOD and LOQ

Signal-to-noise ratios of 3:1 and 10:1 were obtained for LOD and LOQ, respectively. The LOD and LOQ were found to be 51.86 ng/band and 157.17 ng/band for BA and 317.81 ng/spot and 963.07 ng/band for CFC, respectively (Table 2).

3.4.6. Ruggedness and robustness of the method

The standard deviation of peak areas was calculated for each parameter, and the RSD was found to be <2. The low values of the RSD, as shown in Tables 6–9, indicated ruggedness and robustness of the method.

Table 6

Table 7

Table 8

Table 9

The HPTLC–densitometry method for the quantization of BA and CFC is simple, accurate, reproducible and sensitive and is applicable for the analysis of a wide variety of boron-containing dietary supplements. The method was established taking requirements of high precision and economy into consideration [25,27].

The validation parameters for the developed method were the specificity, calibration curve, precision (repeatability), recovery and accuracy. The mobile phase 2-propanol–water 8:2 (v/v) resulted in sharp, symmetrical, and well-resolved peaks at Rf values of 0.58 for CFC and 0.71 for BA, respectively. Linear regression data for the plots confirmed the good linear relationship and the resulting equations were operational in the range of 100–700 ng/band and 500–3500 ng/band, respectively. The method was accurate 98.5–101.8%, with RSD values in the range 1.26–1.89 after spiking the CFC and BA with different concentrations of standard. The current method simultaneously analyze CFC and BA from aqueous extracts of dietary supplements; CFC and BA were identified by comparing their Rf values with those obtained by chromatographic analysis of the reference compounds under the same conditions. The proposed HPTLC–densitometry method can be used for quantitative monitoring of boron in raw materials and prepared formulations without interferences. Its use for standardization and quality control of raw materials and commercial boron products of dietary supplements containing BA/borate and CFC as an ingredient can be explored. Moreover, the detection level is superior to other methods, being suitable for clinical and environmental assays.

4. Conclusion

The HPTLC–densitometry method proposed for the simultaneous quantitative determination of boron as BA and CFC in solid dosage forms is accurate, precise, rapid (30 min. migration time), selective and sensitive. It can, therefore, be easily and conveniently adopted for routine quality control analysis. From an economic point of view, the method used the most simple and cost effective chromatographic technique. Additionally, all of the analytical reagents and solvents are inexpensive and available in any analytical laboratory. Moreover, the method can be also used for boron-dietary supplements monitoring (CFC pharmacokinetics), in order to optimize dosage on an individual basis. The developed method is able to measure the amounts CFC and BA, which can be used in plasma for dose regulation and bioavailability studies, since the method is doing detection and quantification at the ppb level.

Conflict of interests

The authors declare that they have no conflict of interests or financial gains in mentioning the company names or trademarks.

Funding

This work was supported by VDF FutureCeuticals (Momence, IL, USA), University of Medicine and Pharmacy of Craiova and BioBoron Research Institute, Craiova (Romania).

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Table 1. Exact labeling and amounts (mg/tablet or mg/capsule) of the seven boron-dietary supplement products analyzed.

Table 2. Linear regression data for the calibration curve of BA/BX and CFC (n=6).

Table 3. Amount (mg) and percent of free boric acid (BA) and calcium fructoborate complex (CFC) compared with the label value in the seven analyzed products.

Table 4. Precision of the proposed method.

Table 5. Accuracy of the proposed method (n=3).

Table 6. Results from ruggedness studies.

Table 7. Results from robustness studies – mobile phase composition.

Table 8. Results from robustness studies – saturation time.

Table 9. Results from robustness studies – migration distance.

Figure captions

Figure 1. Chemical structure of calcium fructoborate diester.

Figure 2. Densitogram of standard BA (250 ng/band, Rf 0.71±0.02) and standard CFC (1250 ng/band, Rf 0.58±0.02) in 1:1 ratio, obtained at 365 nm.

Figure 3. Chromatograms of the seven boron-dietary supplement products analyzed on a HPTLC silica gel 60 F254 stationary phase by above-described method. The plate was photographed under UV light using a Nikon Coolpix S8000. Lines 1–4: BA standards; Lines 5–8: CFC standards; Lines A–G: Samples.