Application of Liquid Chromatography Coupled to Mass Spectrometry in Quality Assessment of Dietary Supplements—A Case Study of Tryptophan Supplements: Release Assay, Targeted and Untargeted Studies

Dietary supplements are widely consumed in the EU and the USA. Based on their similarity to pharmaceuticals, consumers mistakenly believe that dietary supplements have also been approved for safety and efficacy. However, in the absence of mandatory testing, data on supplement quality is scarce. Thus, we applied liquid chromatography coupled with tandem mass spectrometry to analyse the quality of dietary supplements containing tryptophan (Trp). We examined 22 supplements in tablets or capsules, produced in the USA, Great Britain, Germany, France, Czech Republic, and Poland. Trp release, crucial for bioavailability and efficiency, was assessed. Additionally, we performed a qualitative analysis of the main ingredient and screened for contaminants. Among the contaminants, we detected Trp’s metabolites, condensation products of Trp and carbonyl compounds, Trp degradation products, degradation products of kynurenine, and other contaminants such as glucosamine and melatonin. The main ingredient content was in the range of 55–100% in capsules and 69–87% in tablets. Surprisingly, almost no Trp release was noted from some supplements. Our study confirms the need to advance research on supplements. We believe that the high-quality analysis of supplements based on reliable analytical techniques will be an important contribution to the discussion on the regulatory framework of these products.


Introduction
The consumption of dietary supplements is increasing globally, due to low prices, broad prescription-free distribution, and a common belief in efficacy and safety [1]. In the EU, they are classified as food and contain ingredients with nutritional or physiological effects [2]. Similarly, in the USA, dietary supplements are also classified as food and contain amino acids, herbal substances, vitamins, minerals, and enzymes [3]. Although they are foods, they are sold in typically pharmaceutical dosage forms such as tablets, capsules, sachets, and others designed to be taken in small and defined unit quantities. Based on their similarity to pharmaceuticals, consumers mistakenly believe that dietary supplements have also been approved for safety and efficacy before marketing. However, in the absence of mandatory quality testing, data on supplement quality is scarce. When the data do appear, they indicate some issues such as the presence of contaminants [4,5], the content of the main ingredient lower than the declared one [6,7], or low release from the formulation [8]. The most commonly described contaminants are heavy metals [9], anabolic steroids in preparations for athletes [10], and dioxins in dietary supplements containing fish oil [11]. The amount of the main ingredient in the dietary supplements was only examined for melatonin supplements [1] and supplements containing eicosapentaenoic , Trp degradation products formed after exclusive exposure to heat (c), and reaction products of Trp with aldehydes and ketones. Compounds marked in red were detected in this study [37].
To better visualize the results the heat map was prepared ( Figure 2). We can observe the following: a) Dietary supplements in capsules contained mainly contaminants from group C (Trp degradation products), which may indicate that Trp was obtained by chemical synthesis; b) Dietary supplements in tablets contained mainly contaminants belonging to groups A (Trp's metabolites) and B (condensation products of Trp and carbonyls), which may indicate that Trp was obtained by biotechnology; c) Trp from C1 and C3 dietary supplements might be produced by the same manufacturer. The supplements contained the same contaminants (difference in I3-Trp degradation product, which may be related to different storage conditions);  To summarize, twenty-two compounds were detected in the analysed supplements in the range of 0.02% to 43.89% of the main ingredient area. Among the contaminants, there were Trp's metabolites, condensation products of Trp and carbonyl compounds, Trp degradation products, degradation products of kynurenine, and other contaminants. Some of Trp's contaminants have been already described in Trp raw material of different manufacturers [39], and melatonin supplements [1]. Melatonin can be synthesized from tryptophan by yeast and bacteria, so the occurrence of the contaminants was expected. The biological effect of Trp-related contaminants is unknown. Some Trp degradation products can impact cellular metabolism. I12 a degradation product of Trp was shown to induce apoptosis in a human natural killer cell line. I15 and I17 act as antioxidants and free radical scavengers. However, the dose of I12, I15, I17 needed to have a specific effect on cellular metabolism is unknown. The contaminants were present rather in small amounts, so they may not cause significant side effects [37].
Contamination can occur accidentally, due to poor manufacturing practices or contaminants originating from the supplement ingredients, or intentionally being added by manufacturers. The first group covers heavy metals [9] or substances found in raw materials, e.g., herbicides [43], insecticides [44], mycotoxins [45], and dioxins [11]. All detected contaminants in our study were from this group. Most of them were generated during manufacturing, under storage or transport of supplements/Trp, but some were found in the preparation by accident. Heavy metal analysis was not performed because it requires other analytical techniques such as ICP (inductively coupled plasma) or ASA (atomic absorption analysis). Moreover, these contaminants are mainly detected in herbal-based dietary supplements. Similarly, targeted screening for pesticides and mycotoxins (which were detected in herbal formulations), dioxins (detected in fish oil formulations), cyano- Figure 2. Clustering result of the tested supplements and detected contaminants (using Euclidean distance and clustering algorithm using Ward's method). The level of contaminant is presented as a heatmap (red colour indicates higher concentration and blue colour indicates lower concentration than the average) T-tablet, C-capsule, I-contaminant (with the name of a group of contaminants, i.e., A-Trp's metabolites, B-condensation products of Trp and carbonyls, C-Trp degradation products, D-degradation products of kynurenine, E-other contaminants).
To summarize, twenty-two compounds were detected in the analysed supplements in the range of 0.02% to 43.89% of the main ingredient area. Among the contaminants, there were Trp's metabolites, condensation products of Trp and carbonyl compounds, Trp degradation products, degradation products of kynurenine, and other contaminants. Some of Trp's contaminants have been already described in Trp raw material of different manufacturers [39], and melatonin supplements [1]. Melatonin can be synthesized from tryptophan by yeast and bacteria, so the occurrence of the contaminants was expected. The biological effect of Trp-related contaminants is unknown. Some Trp degradation products can impact cellular metabolism. I12 a degradation product of Trp was shown to induce apoptosis in a human natural killer cell line. I15 and I17 act as antioxidants and free radical scavengers. However, the dose of I12, I15, I17 needed to have a specific effect on cellular metabolism is unknown. The contaminants were present rather in small amounts, so they may not cause significant side effects [37].
Contamination can occur accidentally, due to poor manufacturing practices or contaminants originating from the supplement ingredients, or intentionally being added by manufacturers. The first group covers heavy metals [9] or substances found in raw materials, e.g., herbicides [43], insecticides [44], mycotoxins [45], and dioxins [11]. All detected contaminants in our study were from this group. Most of them were generated during manufacturing, under storage or transport of supplements/Trp, but some were found in the Pharmaceuticals 2022, 15, 448 9 of 27 preparation by accident. Heavy metal analysis was not performed because it requires other analytical techniques such as ICP (inductively coupled plasma) or ASA (atomic absorption analysis). Moreover, these contaminants are mainly detected in herbal-based dietary supplements. Similarly, targeted screening for pesticides and mycotoxins (which were detected in herbal formulations), dioxins (detected in fish oil formulations), cyanobacterial neurotoxins (detected in shark cartilage) and microcystins (detected in algae) because they were not warranted, was not conducted in the study. For instance, pesticides were previously detected in supplements with Ginkgo [44,46] and Ginseng [47,48], whereas mycotoxins were in supplements with Ginkgo and grapes (Table A1). Many of these compounds require targeted screening as well as isolation and enrichment from the complex matrix to obtain a reliable signal [49]. The isolation methods include solid-phase extraction [50], dispersive solid-phase extraction [51], liquid-phase microextraction [52], microwave-assisted extraction [53], microwave-assisted saponification combined with simultaneous unsaponifiable extraction [54]. In our study, a simple extraction was performed without enrichment.
The second group of contaminants includes substances that are prohibited in dietary supplements and are intentionally used by the manufacturer to enhance the observed effect (anabolic steroids [10], hypoglycemic drugs [55], drugs used in potency disorders [56], weight loss products [57]). For Trp supplements, we screened for antidepressants because Trp is often used for depression [28], but no such substances were detected.
The liquid chromatography with mass spectrometry used in this experiment in the untargeted analysis is one of the most frequently used for that purpose. The increased bioavailability of high-resolution instruments improved the detection and identification of compounds in food including dietary supplements. However, confidence in these identifications varies between studies and substances, since it is not always possible or even meaningful to synthesize each substance or confirm them via complementary methods [58]. Thus, we applied the confidence identification level for our data (Table 1). To minimize the risk of false-positive identification it is recommended to search dedicated "small size" MS databases including compounds with a realistic probability to be observed [59]. In our case, the database consisted of degradation products of Trp was used. To decrease further the risk of false-positive identification, all detected compounds were fragmented to achieve a confidence level of at least 3. However, for unexpected compounds such as melatonin or glucosamine, we confirmed the structure with the reference standards. The differences in retention times of these compounds in the samples and reference standard were 0.01 min for glucosamine and 0.03 min for melatonin. The isotopic and fragmentation patterns were similar. The fragmentations are shown in Figure 3.

Determination of Trp in Dietary Supplements
Following the Polish Pharmacopoeia VI, the content of an active substance in tablets or capsules should not exceed the following: (1) ±10% for units with the declared active substance content below 100 mg or (2) ±5% for units with the declared content of the active substance of 100 mg and above. These requirements apply to pharmaceuticals. Due to the lack of specific guidelines for dietary supplements and the fact that dietary supplements appear in the same form as drugs, the same criteria for Trp content were adopted in this study. Therefore, none of the formulations contained the amount of Trp declared by the manufacturer (Table 2), i.e., it was not within 90-110% in each tablet or capsule. The lowest (55% of the declared content) Trp content was in supplement C6, followed by C8 (60%) and T5 (69%). The amount of Trp was within the range of 70-79% in nine supplements and 80-90% in the other nine supplements. The amount of Trp ranged from 70-79% in nine supplements and 80-90% in the other nine supplements. The low Trp content may be due to the lower amount of active ingredients used in production. The highest average Trp content (i.e., 100.45% of the claimed content) was observed in supplement C2. However, the amount of Trp in each capsule varied significantly and ranged from 174 to 251 mg/unit (CV = 19%), indicating improper mixing of the capsule mass. The concentration of the main ingredient in C6 and T2 supplements also had a high coefficient of variation: C6 (CV = 32%), T2 (CV = 35%), but the average amount of Trp was 55% and 87% of the claimed content, respectively. In these cases, both the wrong amount of active ingredient used and improper mixing of the tablet or capsule mass during the manufacturing process may be the reason for inadequate quality.
tifications varies between studies and substances, since it is not always possible or even meaningful to synthesize each substance or confirm them via complementary methods [58]. Thus, we applied the confidence identification level for our data (Table 1). To minimize the risk of false-positive identification it is recommended to search dedicated "small size" MS databases including compounds with a realistic probability to be observed [59]. In our case, the database consisted of degradation products of Trp was used. To decrease further the risk of false-positive identification, all detected compounds were fragmented to achieve a confidence level of at least 3. However, for unexpected compounds such as melatonin or glucosamine, we confirmed the structure with the reference standards. The differences in retention times of these compounds in the samples and reference standard were 0.01 min for glucosamine and 0.03 min for melatonin. The isotopic and fragmentation patterns were similar. The fragmentations are shown in Figure 3. melatonin glucosamine     Inconsistency between the declared and determined content of the main ingredient has been previously reported for melatonin supplements [1] and lutein [8]. None of the lutein supplements (n = 10) and 41% of the melatonin supplements (n = 17) met our criteria. However, it is not clear whether the melatonin or lutein content was evenly distributed among the units. Therefore, no conclusions could be drawn regarding quality.
Content uniformity is an important critical quality attribute. High variability in active ingredient content can be caused by the following: improper particle distribution (e.g., agglomeration); poor macro-and microblending at the powder mixing stage; loss of a component (e.g., due to adsorption to the equipment surface); thief sampling and analytical errors; segregation of well-mixed blends during powder transfer, handling or further operations [60]. The controlling of all this process is required. In the case of pharmaceuticals available on the market, content uniformity is not likely to occur [61,62]. The only study showing the problem with this attribute concern tablets splitting [63]. However, a large number of articles on content uniformity and the ways of continuous monitoring tablet content uniformity [64] suggest that it is a difficult task to achieve.

Dissolution Test for Trp Tablets and Capsules
The Food and Drug Administration provides guidelines for drug testing. According to the dissolution test requirements, the active ingredient should release from the immediaterelease oral solid drug at least 80% of its claimed content after 30 min of the release test [65].
Dietary supplements do not have dedicated guidelines for dissolution testing. Therefore, the same criteria for the dissolution test were used in this study. Trp release higher than 80% was determined for supplements C6, C7, C10 at pH 1.2 and supplement T9, T10 at pH 6.8. (Table 3, Figure 4). Supplement C11 had the lowest release (1.22%) at gastric pH (pH 1.2), while at pH 6.8 the release reached 60.2%. Supplements C2 and C3 were characterized by a release of no more than 5% Trp at both pHs (1.2 and 6.8). Thus, only up to 5% of the claimed dose of Trp could be absorbed from these supplements across biological membranes to produce a physiological effect (Figure 4a). Trp release between 10 and 20% regardless of dissolution medium was determined for C8 and between 20% and 30% for T3 and T7. In summary, Trp release for 10 of the 22 supplements was determined between 1.22 and 59.9% at both pHs ( Figure 4).  Trp was completely released from C6, C10, T1, T2, T11 supplements at both pHs (Table 3, Figure 4). The other five supplements released Trp at only one pH: gastric (supplement C7, C9) or intestinal (supplement C1, C11, T4, T9). Thus, in these supplements, the amount of Trp released was limited only by the content of the main ingredient (Table 2). No negative effect of technological parameters and excipients was observed. One of these supplements, i.e., C11 was probably designed by the manufacturer as an enteral form, which was not even mentioned in the packaging. For this supplement, a release of less than 10% was observed at pH 1.2 (as recommended by the guidelines) and a complete release was observed at pH 6.8. However, due to the lower Trp content, the complete release did not reach 80% of the claimed content as recommended.
The low release of Trp from C2 (pH = 1.2, release 2.65%; pH = 6.8, release 2.3%) and C3 (pH = 1.2, release 3.08%; pH = 6.8, release 4.8%) was mainly due to improperly selected process parameters and/or improperly selected excipients. This is because the content of Trp in the dosage form was much higher than the amount of Trp released (Figure 4a). In the remaining formulations (i.e., C4, C6, C8, C9, C11, T1, T4, T5, T6, T9), the low Trp release was due to both low compound content in the formulation and inappropriate preparation technology (poorly selected technological parameters or excipients) (Figure 4). Referring to in vivo conditions, units characterized by low release will enter the gastric juice but will not release the substance. Thus, no physiological effect will be observed.   In summary, none of the analysed dietary supplements contained 80% or more Trp (for each tablet/capsule), which means that these formulations do not meet the release requirements for medicinal products. Comparative data on Trp release from other dietary supplements are not available. Applying our criteria to dietary supplements containing lutein [8], it also the case that none of these dietary supplements would meet these requirements. However, a comparison between the two studies is not easy because the release of the lutein supplements was performed using unconventional parameters. The dissolution test fluid for tablets was 2% P80 (w/v) and for capsules 2% P80 (w/v) with 25% ethanol. In the case of Trp supplements, the release test fluid was 0.1 mol/L hydrochloric acid (simulated gastric conditions) and 0.05 mol/L phosphate buffer (pH 6.8, simulated intestinal conditions) regardless of the form of the dietary supplement. Low release of active ingredients such as calcium carbonate [13], melatonin [14], folic acid [15,16], iron, zinc, manganese [17] and Grape seed extract [19], have also been observed in other dietary supplements in solid form (Table A2). However, in these cases, the reasons for the low release are not known due to the lack of data on the content of the main ingredient in these supplements. Low release may be due to insufficient content of the main ingredient, improperly selected process parameters, and/or improperly selected excipients. Only for food supplements containing triiodothyronine (n = 3) or prehormone thyroxine (n = 1) was the main component release above 93% [18].
In our study, Trp release was higher from tablets (12.1-81.4%) than from capsules (1.22-90.4%). Similar results were previously obtained for lutein [8] and folic acid [16]. For lutein supplements, release from capsules (made in the USA), despite containing adequate amounts of lutein, showed alarming results due to poor dissolution properties (less than 20% after 180 min of testing). These results may contribute to the lack of bioavailability of lutein. Unlike the capsules, the lutein tablets (made in Brazil) released more than 80% of the lutein within 180 min.
Analysis of the content, identity, and release of active ingredients from products is important to assess their quality [66]. The dissolution test determines the amount of active substance released and is mandatory for solid drug forms, but not for the same forms of dietary supplements [67]. The in vivo absorption of the active ingredient from solid formulations can be predicted to some extent using this assay [68]. A low release rate means low absorption and no intended effect. Thus, even the substance is in labelled amounts in the supplement but is not released, the consumer will not be able to achieve the effect.
Ease of marketing the supplement and low level of control combined with high popularity and high market value make dietary supplements a group of products particularly vulnerable to negligence or intentional manipulation, which poses a threat to consumers' interests and sometimes even their health [21,69,70]. Determining the quality of dietary supplements is challenging and can be more difficult than for pharmaceutical products because such products often contain multiple vitamins, minerals [71], many of which are derived from plants [72] or other biological sources [73]. However, quality control of supplements should meet the same standards as pharmaceutical products because in both cases they are intended for consumer use [74]. The results of our studies developed with the use of a gold standard in analytics-mass spectrometry coupled with liquid chromatography, provide important data on the quality of the analysed dietary supplements. We hope that our results will encourage further research and increase public awareness about the purposefulness and safety of taking dietary supplements. An informed consumer will choose tested supplements, which will encourage manufacturers to test. In the case of the Food and Drug Administration, Good Manufacturing Practice in Manufacturing, Packing, Labelling, or Holding Operations for Dietary Supplements were already established. Applying GMPs to dietary supplements would be a further step to ensure products are consistently produced and controlled to the quality standards appropriate to their intended use [74].
A limitation of our study is the inability to detect compounds present at very low concentrations. These compounds require appropriate sample preparation. In addition, the targeted analysis should be chosen over non-targeted screening in their case.

Samples
The study was conducted on twenty-two Trp supplements, which is 10% of all dietary supplements with Trp in tablets or capsules registered in Poland, and all available on the market. There were two types of dosage forms: capsules (C1-C12) and tablets (T1-T10). All supplements were manufactured in the EU (Poland, UK, France, Germany, Czech Republic) and the USA. Six supplements were purchased in a Polish online e-commercial platform, the rest in pharmacies or online pharmacies in Poland.

Sample Preparation
Three tablets or capsules were randomly selected from each supplement. The total weight of three tablets, or of the contents of three capsules, were determined. For tablets, a grinding step was applied. In the next step, the tablet's mass or capsule content equivalent to 10 mg Trp was weighed and 1.00 mL of acetonitrile/methanol/water (1:1:1; v/v/v) mixture was added. The mixture was sonicated for 15 min and centrifuged for 5 min. The supernatant was then diluted with mobile phase to a concentration of 500 ng/mL or 100 ng/mL for qualitative and quantitative analysis, respectively. For quantitative analysis, an internal standard (doxepin) was added in the last step to a final concentration of 500 ng/mL.

Qualitative Analysis
Instrumental analysis was performed using a UHPLC Dionex Ultimate 3000 with a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer system equipped with heat electrospray ionization (HESI), an online vacuum degasser, a quaternary pump, an autosampler, and a thermostatted column compartment. The HESI was operated in positive mode. Full MS scans were acquired over the m/z 100-1400 range with a resolution of 70,000 (m/z 200). Fragmentation was performed in different runs with a normalized collision energy of 20, 35, 50 eV. The ion selection threshold was 8 × 10 3 counts, and the maximum allowed ion accumulation times were set to auto both for full MS scans and for the tandem mass spectrum. Standard mass spectrometric conditions for all experiments were: spray voltage, 3.5 kV; sheath gas pressure: 60 arb; aux gas pressure: 20 arb; sweep gas pressure: 0 arb, heated capillary temperature: 320 • C; loop count: 3; isolation window: m/z 1.0; and dynamic exclusion: 6.0 s. For all full scan measurements, lock-mass ions from ambient air (m/z 445.1200 and 291.2842) were used as internal calibrants.
Chromatographic separation was achieved with an Accucore C-18 column (100 mm × 4.6 mm, 2.6 µm) supplied by Thermo Fisher Scientific (Waltham, MA, USA) equipped with a security guard. The column was maintained at 40 • C at a flow rate of 0.3 mL/min. The mobile phases consisted of HPLC grade water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as eluent B. The gradient (% B) was as follows: 0 min 10%; 1 min 10%; 10 min 95%; 15 min 95%. The volume of injection was 10 µL.
The results obtained were analysed using Compound Discoverer 3.0 software supplied by Thermo Fisher Scientific (Waltham, MA, USA).
The structures of the metabolites were proposed based on: 1.
The m/z of the compound. The difference between experimental and theoretical molecular weight should be no higher than 5 ppm; 2.
The isotopic pattern. The relative intensity tolerance to be used for the isotope search was set at 30%; 3.
Fragmentation of the compound. The fragmentation spectrum was compared with experimental data found in the mass spectra library or the literature (confidence level 2), in silico fragmentation (confidence level 3) or reference standard (confidence level 1).

Quantitative Analysis
The instrumental analysis was performed using an Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA), equipped with a degasser, autosampler, and binary pump coupled to a QTRAP 4000 hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex, Framingham, MA, USA). The Turbo Ion Spray source was operated in positive mode. The curtain gas, ion source gas 1, ion source gas 2, and collision gas (all high-purity nitrogen) were set at 0.24 MPa, 0.41 MPa, 0.28 MPa, and "medium" instrument units, respectively. The ion spray voltage and source temperature were 4500 V and 600 • C, respectively. The target compounds were analysed in multiple reaction monitoring (MRM) mode. The compounds parameters, viz. declustering potential (DP), collision energy (CE), entrance potential (EP), and collision exit potential (CXP), were 76, 27, 12 V and 71, 25, 14 V for Trp and doxepin, respectively.
The analysis of the Trp content in dietary supplements was preceded by method validation. The parameters tested were selectivity, precision, accuracy, linearity, and limit of quantification. The range of the calibration curve was selected as 0.01-10 µg/mL. Accuracy and precision were determined in triplicate at four concentration levels (0.01, 0.05, 5.0 and 10.0 µg/mL).
Calculations were made using the Analyst 1.6.3 software (AB Sciex, Framingham, MA, USA).

Dissolution Test for Tablets or Capsules
Trp release study was performed using a USP II Varian VK 7025 or USP I Varian VK 7025 dissolution tester (Erweka GmbH, Heusenstamm, Germany) for tablets and capsules, respectively. Six tablets or capsules were randomly selected and individually placed in the dissolution vessels. Each vessel contained 900 mL of dissolution medium. The stirring speed of 50 rpm or 100 rpm was used for tablets and capsules, respectively. The temperature was set at 37 ± 0.5 • C. Aliquots (1.5 mL) of the medium were manually collected using 5 mL syringes after 30 min of the test and filtered through a Millex-HA 0.45 µm filter. Each aliquot withdrawn was replaced with 1.5 mL of fresh medium. The experiment was performed both in hydrochloride acid pH 1.2 (simulated gastric conditions) and phosphate buffer pH 6.8 (simulated intestinal conditions). The Trp content was measured as described in Section 3.5 (Quantitative analysis).

Expanded Uncertainty
To assess whether the amount of Trp in the dosage unit and amount of the compound released is equal within the uncertainty range, extended uncertainty was determined using Equation (1).
The measurement results were equal if: x 1 -mean [mg] Trp content determined in dosage unit using quantitative analysis (n = 3). n-the number of tablets or capsules analysed.

Conclusions
A new analytical approach based on liquid chromatography coupled to mass spectrometry provided the opportunity to obtain reliable results on the quality of dietary supplements. The quality of supplements is lower than that of pharmaceuticals with lower than claimed amounts of the main ingredient and a lack of uniform distribution between units. Sometimes, the release of the main ingredient is low, resulting in a lower probability of absorption and physiological effect. Contaminants were detected in all dietary supplements analysed, based on untargeted analysis. These substances, in the amounts determined, may not affect health or show significant unknown effects. The study confirms issues with the quality of dietary supplements and provides an important contribution to the discussion on the regulation of dietary supplements. We believe that the new analytical approach will have broad applicability in the assessment of supplement quality.

Acknowledgments:
The authors express their sincere gratitude to Anna Harasimiuk and Jakub Niewiarowski for their help in performing the Trp dissolution test in pH 1.2, which was part of their master thesis.

Acknowledgments:
The authors express their sincere gratitude to Anna Harasimiuk and Jakub Niewiarowski for their help in performing the Trp dissolution test in pH 1.2, which was part of their master thesis.

Conflicts of Interest:
The authors declare no conflict of interest.