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Javier Fidalgo, * Pierre-Antoine Deglesne, * Rodrigo Arroyo, * Lilian Sepúlveda, * Evgeniya Ranneva, Philippe Deprez Department of Science, Skin Tech Pharma Group, Castello D’Empúries, Catalonia, Spain * These authors have some insights into this work Equal contribution background: Hyaluronic acid (HA) is a naturally occurring polysaccharide used in the production of dermal fillers for aesthetic purposes. Since it has a half-life of several days in human tissues, HA-based dermal fillers are chemically modified to extend their life in the body. The most common modification in commercial HA-based fillers is the use of 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking agent to crosslink the HA chains. Residual or unreacted BDDE is considered non-toxic at <2 parts per million (ppm); therefore, the residual BDDE in the final dermal filler must be quantified to ensure patient safety. Materials and methods: This study describes the detection and characterization of a by-product of the cross-linking reaction between BDDE and HA under alkaline conditions by combining liquid chromatography and mass spectrometry (LC-MS). Results: After different analyses, it was found that the alkaline conditions and high temperature used to disinfect the HA-BDDE hydrogel promoted the formation of this new by-product, the “propylene glycol-like” compound. LC-MS analysis confirmed that the by-product has the same monoisotopic mass as BDDE, a different retention time (tR), and a different UV absorbance (λ=200 nm) mode. Unlike BDDE, it was observed in LC-MS analysis that under the same measurement conditions, this by-product has a higher detection rate at 200 nm. Conclusion: These results indicate that there is no epoxide in the structure of this new compound. The discussion is open to assess the risk of this new by-product found in the production of HA-BDDE hydrogel (HA dermal filler) for commercial purposes. Keywords: hyaluronic acid, HA dermal filler, cross-linked hyaluronic acid, BDDE, LC-MS analysis, BDDE by-product.
Fillers based on hyaluronic acid (HA) are the most common and popular dermal fillers used for cosmetic purposes. 1 This dermal filler is a hydrogel, usually composed of >95% water and 0.5-3% HA, which gives them a gel-like structure. 2 HA is a polysaccharide and the main component of the extracellular matrix of vertebrates. One of the ingredients. It consists of (1,4)-glucuronic acid-β (1,3)-N-acetylglucosamine (GlcNAc) repeating disaccharide units connected by glycosidic bonds. This disaccharide pattern is the same in all organisms. Compared with some protein-based fillers (such as collagen), this property makes HA a highly biocompatible molecule. These fillers can exhibit amino acid sequence specificity that may be recognized by the patient’s immune system.
When used as a dermal filler, the main limitation of HA is its rapid turnover within the tissues due to the presence of a specific family of enzymes called hyaluronidases. So far, several chemical modifications in the HA structure have been described to increase the half-life of HA in tissues. 3 Most of these modifications attempt to reduce the access of hyaluronidase to polysaccharide polymers by cross-linking HA chains. Therefore, due to the formation of bridges and the intermolecular covalent bonds between the HA structure and the cross-linking agent, the cross-linked HA hydrogel produces more anti-enzyme degradation products than the natural HA. 4-6
So far, the chemical crosslinking agents used to produce crosslinked HA include methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) And poly(ethylene glycol) diglycidyl ether. 10 ,11 BDDE is currently the most commonly used crosslinking agent. Although these types of hydrogels have been proven to be safe for decades, the crosslinking agents used are reactive reagents that may be cytotoxic and, in some cases, mutagenic. 12 Therefore, their residual content in the final hydrogel must be high. BDDE is considered safe when the residual concentration is less than 2 parts per million (ppm). 4
There are several methods to detect low-residue BDDE concentration, cross-linking degree and substitution position in HA hydrogels, such as gas chromatography, size exclusion chromatography coupled with mass spectrometry (MS), nuclear magnetic resonance (NMR) fluorescence measurement methods, and Diode array coupled high performance liquid chromatography (HPLC). 13-17 This study describes the detection and characterization of a by-product in the final cross-linked HA hydrogel produced by the reaction of BDDE and HA under alkaline conditions. HPLC and liquid chromatography-mass spectrometry (LC-MS analysis). Since the toxicity of this by-product of BDDE is unknown, we recommend that its residue quantification should be determined in a similar manner to the method usually performed on BDDE in the final product.
The obtained sodium salt of HA (Shiseido Co., Ltd., Tokyo, Japan) has a molecular weight of ~1,368,000 Da (Laurent method) 18 and an intrinsic viscosity of 2.20 m3/kg. For the crosslinking reaction, BDDE (≥95%) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Phosphate buffered saline with pH 7.4 was purchased from Sigma-Aldrich Company. All solvents, acetonitrile and water used in the LC-MS analysis were purchased from HPLC grade quality. Formic acid (98%) is purchased as reagent grade.
All experiments were performed on a UPLC Acquity system (Waters, Milford, MA, USA) and connected to an API 3000 triple quadrupole mass spectrometer equipped with an electrospray ionization source (AB SCIEX, Framingham, MA, USA).
The synthesis of cross-linked HA hydrogels was started by adding 198 mg of BDDE to a 10% (w/w) sodium hyaluronate (NaHA) solution in the presence of 1% alkali (sodium hydroxide, NaOH). The final BDDE concentration in the reaction mixture was 9.9 mg/mL (0.049 mM). Then, the reaction mixture was thoroughly mixed and homogenized and allowed to proceed at 45°C for 4 hours. 19 The pH of the reaction is maintained at ~12.
Thereafter, the reaction mixture was washed with water, and the final HA-BDDE hydrogel was filtered and diluted with PBS buffer to achieve a HA concentration of 10 to 25 mg/mL, and a final pH of 7.4. In order to sterilize the produced cross-linked HA hydrogels, all these hydrogels are autoclaved (120°C for 20 minutes). The purified BDDE-HA hydrogel is stored at 4°C until analysis.
To analyze the BDDE present in the cross-linked HA product, a 240 mg sample was weighed and introduced into the center hole (Microcon®; Merck Millipore, Billerica, MA, USA; volume 0.5 mL) and centrifuged at 10,000 rpm at room temperature 10 minute. A total of 20 µL of pull-down liquid was collected and analyzed.
In order to analyze the BDDE standard (Sigma-Aldrich Co) under alkaline conditions (1%, 0.1% and 0.01% NaOH), if the following conditions are met, the liquid sample is 1:10, 1:100, or up to 1:1,000,000 If necessary, use MilliQ deionized water for analysis.
For the starting materials used in the cross-linking reaction (HA 2%, H2O, 1% NaOH, and 0.049 mM BDDE), 1 mL of each sample prepared from these materials was analyzed using the same analysis conditions.
To determine the specificity of the peaks appearing in the ion map, 10 µL of 100 ppb BDDE standard solution (Sigma-Aldrich Co) was added to the 20 µL sample. In this case, the final concentration of the standard in each sample is 37 ppb.
First, prepare a BDDE stock solution with a concentration of 11,000 mg/L (11,000 ppm) by diluting 10 μL of standard BDDE (Sigma-Aldrich Co) with 990 μL MilliQ water (density 1.1 g/mL). Use this solution to prepare a 110 µg/L (110 ppb) BDDE solution as an intermediate standard dilution. Then, use the intermediate BDDE standard diluent (110 ppb) to prepare the standard curve by diluting the intermediate diluent several times to achieve the desired concentration of 75, 50, 25, 10, and 1 ppb. As shown in Figure 1, it is found that the BDDE standard curve from 1.1 to 110 ppb has good linearity (R2>0.99). The standard curve was repeated in four independent experiments.
Figure 1 BDDE standard calibration curve obtained by LC-MS analysis, in which a good correlation is observed (R2>0.99).
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; LC-MS, liquid chromatography and mass spectrometry.
In order to identify and quantify the BDDE standards present in the cross-linked HA and the BDDE standards in the base solution, LC-MS analysis was used.
The chromatographic separation was achieved on a LUNA 2.5 µm C18(2)-HST column (50×2.0 mm2; Phenomenex, Torrance, CA, USA) and kept at room temperature (25°C) during the analysis. The mobile phase consists of acetonitrile (solvent A) and water (solvent B) containing 0.1% formic acid. The mobile phase is eluted by gradient elution. The gradient is as follows: 0 minutes, 2% A; 1 minute, 2% A; 6 minutes, 98% A; 7 minutes, 98% A; 7.1 minutes, 2% A; 10 minutes , 2% A. The running time is 10 minutes and the injection volume is 20 µL. The retention time of BDDE is about 3.48 minutes (ranging from 3.43 to 4.14 minutes based on experiments). The mobile phase was pumped at a flow rate of 0.25 mL/min for LC-MS analysis.
For BDDE analysis and quantification by MS, the UPLC system (Waters) is combined with an API 3000 triple quadrupole mass spectrometer (AB SCIEX) equipped with an electrospray ionization source, and the analysis is performed in positive ion mode (ESI+).
According to the ion fragment analysis performed on BDDE, the fragment with the highest intensity was determined to be the fragment corresponding to 129.1 Da (Figure 6). Therefore, in the multi-ion monitoring mode (MIM) for quantification, the mass conversion (mass-to-charge ratio [m/z]) of BDDE is 203.3/129.1 Da. It also uses full scan (FS) mode and product ion scan (PIS) mode for LC-MS analysis.
In order to verify the specificity of the method, a blank sample (initial mobile phase) was analyzed. No signal was detected in the blank sample with a mass conversion of 203.3/129.1 Da. Regarding the repeatability of the experiment, 10 standard injections of 55 ppb (in the middle of the calibration curve) were analyzed, resulting in a residual standard deviation (RSD) <5% (data not shown).
The residual BDDE content was quantified in eight different autoclaved BDDE cross-linked HA hydrogels, corresponding to four independent experiments. As described in the “Materials and Methods” section, the quantification is evaluated by the average value of the regression curve of the BDDE standard dilution, which corresponds to the unique peak detected at the BDDE mass transition of 203.3/129.1 Da, with a retention time of 3.43 to 4.14 minutes Not waiting. Figure 2 shows an example chromatogram of the 10 ppb BDDE reference standard. Table 1 summarizes the residual BDDE content of eight different hydrogels. The value range is 1 to 2.46 ppb. Therefore, the residual BDDE concentration in the sample is acceptable for human use (<2 ppm).
Figure 2 Ion chromatogram of 10 ppb BDDE reference standard (Sigma-Aldrich Co), MS (m/z) transition obtained by LC-MS analysis of 203.30/129.10 Da (in positive MRM mode).
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring; MS, mass; m/z, mass-to-charge ratio.
Note: Samples 1-8 are autoclaved BDDE cross-linked HA hydrogels. The residual amount of BDDE in the hydrogel and the peak of BDDE retention time are also reported. Finally, the existence of new peaks with different retention times is also reported.
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; HA, hyaluronic acid; MRM, multiple reaction monitoring; tR, retention time; LC-MS, liquid chromatography and mass spectrometry; RRT, relative retention time.
Surprisingly, the analysis of the LC-MS ion chromatogram showed that based on all the autoclaved cross-linked HA hydrogel samples analyzed, there was an extra peak at the shorter retention time of 2.73 to 3.29 minutes. For example, Figure 3 shows the ion chromatogram of a cross-linked HA sample, where an additional peak appears at a different retention time of approximately 2.71 minutes. The observed relative retention time (RRT) between the newly observed peak and the peak from BDDE was found to be 0.79 (Table 1). Since we know that the newly observed peak is less retained in the C18 column used in the LC-MS analysis, the new peak may correspond to a more polar compound than BDDE.
Figure 3 Ion chromatogram of cross-linked HA hydrogel sample obtained by LC-MS (MRM mass conversion 203.3/129.0 Da).
Abbreviations: HA, hyaluronic acid; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring; RRT, relative retention time; tR, retention time.
In order to rule out the possibility that the new peaks observed may be contaminants originally present in the raw materials used, these raw materials were also analyzed using the same LC-MS analysis method. The starting materials analyzed include water, 2% NaHA in water, 1% NaOH in water, and BDDE at the same concentration used in the cross-linking reaction. The ion chromatogram of the starting material used did not show any compound or peak, and its retention time corresponds to the new peak observed. This fact discards the idea that not only the starting material may contain any compounds or substances that may interfere with the analysis procedure, but there is no sign of possible cross-contamination with other laboratory products. The concentration values obtained after LC-MS analysis of BDDE and new peaks are shown in Table 2 (samples 1-4) and the ion chromatogram in Figure 4.
Note: Samples 1-4 correspond to the raw materials used to produce autoclaved BDDE cross-linked HA hydrogels. These samples were not autoclaved.
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; HA, hyaluronic acid; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring.
Figure 4 corresponds to the LC-MS chromatogram of a sample of the raw material used in the cross-linking reaction of HA and BDDE.
Note: All of these are measured at the same concentration and ratio used to carry out the cross-linking reaction. The numbers for the raw materials analyzed by the chromatogram correspond to: (1) water, (2) 2% HA aqueous solution, (3) 1% NaOH aqueous solution. LC-MS analysis is performed for mass conversion of 203.30/129.10 Da (in positive MRM mode).
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; HA, hyaluronic acid; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring.
The conditions that led to the formation of new peaks were studied. In order to study how the reaction conditions used to produce the cross-linked HA hydrogel affect the reactivity of the BDDE cross-linking agent, leading to the formation of new peaks (possible by-products), different measurements were performed. In these determinations, we studied and analyzed the final BDDE crosslinker, which was treated with different concentrations of NaOH (0%, 1%, 0.1%, and 0.01%) in an aqueous medium, followed by or without autoclaving. The bacteria procedure to simulate the same conditions is the same as the method used to produce the cross-linked HA hydrogel. As described in the “Materials and Methods” section, the mass transition of the sample was analyzed by LC-MS to 203.30/129.10 Da. The BDDE and the concentration of the new peak are calculated, and the results are shown in Table 3. No new peaks were detected in the samples that were not autoclaved, regardless of the presence of NaOH in the solution (samples 1-4, Table 3). For autoclaved samples, new peaks are only detected in the presence of NaOH in the solution, and the formation of the peak seems to depend on the NaOH concentration in the solution (samples 5-8, Table 3) (RRT = 0.79). Figure 5 shows an example of an ion chromatogram, showing two autoclaved samples in the presence or absence of NAOH.
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring.
Note: The top chromatogram: The sample was treated with 0.1% NaOH aqueous solution and autoclaved (120°C for 20 minutes). Bottom chromatogram: The sample was not treated with NaOH, but autoclaved under the same conditions. The mass conversion of 203.30/129.10 Da (in positive MRM mode) was analyzed by LC-MS.
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring.
In all autoclaved samples, with or without NaOH, the BDDE concentration was greatly reduced (up to 16.6 times) (samples 5-8, Table 2). The decrease in BDDE concentration may be due to the fact that at high temperatures, water can act as a base (nucleophile) to open the epoxide ring of BDDE to form a 1,2-diol compound. The monoisotopic quality of this compound is different from that of BDDE and therefore will not be affected. LC-MS detected a mass shift of 203.30/129.10 Da.
Finally, these experiments show that the generation of new peaks depends on the presence of BDDE, NAOH, and the autoclaving process, but has nothing to do with HA.
The new peak found at a retention time of approximately 2.71 minutes was then characterized by LC-MS. For this purpose, BDDE (9.9 mg/mL) was incubated in a 1% NaOH aqueous solution and autoclaved. In Table 4, the characteristics of the new peak are compared with the known BDDE reference peak (retention time approximately 3.47 minutes). Based on the ion fragmentation analysis of the two peaks, it can be concluded that the peak with a retention time of 2.72 minutes shows the same fragments as the BDDE peak, but with different intensities (Figure 6). For the peak corresponding to the retention time (PIS) of 2.72 minutes, a more intense peak was observed after fragmentation at a mass of 147 Da. At the BDDE concentration (9.9 mg/mL) used in this determination, different absorbance modes (UV, λ=200 nm) in the ultraviolet spectrum were also observed after chromatographic separation (Figure 7). The peak with a retention time of 2.71 minutes is still visible at 200 nm, while the BDDE peak cannot be observed in the chromatogram under the same conditions.
Table 4 Characterization results of the new peak with a retention time of about 2.71 minutes and the BDDE peak with a retention time of 3.47 minutes
Note: To obtain these results, LC-MS and HPLC analyses (MRM and PIS) were performed on the two peaks. For HPLC analysis, UV detection with a wavelength of 200 nm is used.
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring; m/z, mass-to-charge ratio; PIS, product Ion scanning; ultraviolet light, ultraviolet light.
Note: The mass fragments are obtained by LC-MS analysis (PIS). Top chromatogram: mass spectrum of BDDE standard sample fragments. Bottom chromatogram: The mass spectrum of the new peak detected (RRT associated with the BDDE peak is 0.79). BDDE was processed in 1% NaOH solution and autoclaved.
Abbreviations: BDDE, 1,4-butanediol diglycidyl ether; LC-MS, liquid chromatography and mass spectrometry; MRM, multiple reaction monitoring; PIS, product ion scan; RRT, relative retention time.
Figure 7 Ion chromatogram of the 203.30 Da precursor ion, and (A) the new peak with a retention time of 2.71 minutes and (B) the UV detection of the BDDE reference standard peak at 3.46 minutes at 200 nm.
In all the cross-linked HA hydrogels produced, it was observed that the residual BDDE concentration after LC-MS quantification was <2 ppm, but a new unknown peak appeared in the analysis. This new peak does not match the BDDE standard product. The BDDE standard product has also undergone the same quality conversion (MRM conversion 203.30/129.10 Da) analysis in the positive MRM mode. Generally, other analytical methods such as chromatography are used as limit tests to detect BDDE in hydrogels, but the maximum detection limit (LOD) is slightly lower than 2 ppm. On the other hand, so far, NMR and MS have been used to characterize the degree of cross-linking and/or modification of HA in the sugar unit fragments of cross-linked HA products. The purpose of these techniques has never been to quantify residual BDDE detection at such low concentrations as we describe in this article (LOD of our LC-MS method = 10 ppb).
Post time: Sep-01-2021