Derivatizing Agent Selection for Hydrophilic Lysine- and Arginine-Containing Tetradecapeptide Analysis in Human Plasma by RP HPLC-MS/MS

Abstract

The application of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) in the analysis of peptide therapeutics demonstrates its capacity to achieve high sensitivity and selectivity, which are essential qualities for the expanding peptide therapeutic industry. Given the challenges posed by hydrophilic peptides in reversed-phase chromatography, we investigated the necessity of a derivatization procedure to improve chromatographic separation and quasimolecular ion fragmentation during MS/MS detection. We investigated how eight different derivatizing agents react with a hydrophilic lysine- and arginine-containing human ezrin peptide-1 (HEP-1) to identify the most suitable one. The results showed that the reaction of HEP-1 with propionic anhydride proceeds most rapidly and completely, providing a high and reproducible yield of the product, which has sufficient retention on the RP column. The 4-propionylated derivative of HEP-1, compared to the other derivatives considered, demonstrates the most pronounced MS/MS fragmentation. The retention time of 2.42 min allows the separation of the substance from the interfering components of the blood plasma matrix and provides a limit of quantification of 5.00 ng/mL, which allows the use of this derivatizing agent for subsequent applications in pharmacokinetic studies, and this approach can improve the analytical parameters of similar peptides in other HPLC-MS/MS studies.

1. Introduction

The study of peptides is currently at the cutting edge of research interest. The scientific community is primarily involved in two distinct areas of investigation: proteomics and peptide drug discovery.

Proteomic research aims to study the structure, functions, and interactions of proteins in biological systems and their role in cellular processes. Thus, the research is relevant to both fundamental and applied research, such as clinical trials [1]. Peptide drug discovery focuses on improving the efficacy of the peptides used, for example, by searching for alternative routes of administration [2], increasing membrane permeability, oral bioavailability, and in vivo stability [3].

In both proteomics and peptide drug discovery, there is a need to identify and quantify peptides, for which there are various techniques, including immunological methods, such as radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry (MS) methods [4]. The latter, especially high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS), has found the greatest application [5,6].

Proteomics has two main approaches: top-down and bottom-up [7]. In top-down proteomics, whole proteins are analyzed by a matrix-assisted laser desorption ionization source (MALDI)-MS/MS to identify and characterize, whereas in bottom-up proteomics, enzymatic hydrolysis is performed. The resulting peptides are then separated and analyzed by HPLC-MS/MS [8].

For peptide therapeutics, HPLC-MS/MS is a powerful technique with applications in many areas, starting with peptide characterization [6] and quality control [9], followed by investigation in clinical trials [10,11]. Quantification for pharmacokinetic studies is of special concern because therapeutic peptides are known for their high potency at low doses [12], which makes analysis difficult due to low concentrations in biological samples, such as blood [13]. Although HPLC-MS/MS can be considered a highly sensitive and selective method [14], additional difficulties may occur.

A peptide is a chain of up to 40 amino acids that vary in structure and properties [12,15]. Among the variety of amino acids, lysine and arginine are of particular interest. Lysine- and arginine-containing peptides have great potential as cell-penetrating peptides (CPPs) for delivery vectors [16]. In addition, peptides enriched with these amino acids tend to exhibit antimicrobial properties [17,18]. Such peptides show high hydrophilicity, which can be a challenge during chromatographic separation in reversed-phase (RP) HPLC. In addition, the fragmentation of such peptides may be difficult and requires increased collision energy due to charge localization on basic groups [19]. For some of these peptides, hydrophilic interaction liquid chromatography (HILIC) is an option [20,21]. However, it does not improve the fragmentation properties. In such a case, derivatization is often used to solve both problems [10,22,23].

Derivatization-based HPLC-MS/MS is a highly effective tool for solving many analytical problems and greatly expanding the applications of HPLC-MS/MS in various research areas [23,24]. Derivatization methods are widely used for quantitative proteomics analysis, such as an isotope-coded affinity tag (ICAT) or isotope-coded protein label (ICPL), isobaric tag for relative and absolute quantitation (iTRAQ), and tandem mass tag (TMT) [22]. There are multiple derivatization methods for the analysis of active compounds in peptide drug research studies [25,26,27,28]. Despite growing interest, the literature currently lacks comprehensive and comparative studies on the derivatization of hydrophilic peptides for enhanced analytical performance. Nevertheless, derivatization-based HPLC-MS/MS has become an important tool for peptide analysis [23].

Despite the need for additional reaction steps, derivatization have been considered by many scientists as a last resort to overcome detection and occasional separation problems, but additional matrix effects assessment is essential due to the presence of interference caused by excess reagents and byproducts [29].

The determination of lysine- and arginine-rich peptides in biological matrices by RP HPLC-MS/MS is quite challenging. The pronounced hydrophilicity and basicity of the human ezrin peptide-1 (HEP-1), in which we have chosen a lysine- and arginine-containing peptide as an example, result in substantial difficulties when employing RP HPLC-MS/MS. It is demanding to achieve optimal conditions for separation. In addition, the choice of fragmentation conditions can be rather energy- and time-consuming. Therefore, though a derivatization process can be desirable for analytical scientists, there is still a need for an appropriate choice regarding the derivatization reagent, which can be laborious.

The aim of the present study is to provide a step-by-step derivatizing agent selection for hydrophilic lysine- and arginine-containing HEP-1 for further analysis by HPLC-MS/MS in human plasma.

2. Results and Discussion

2.1. Peptide Therapeutic to Study

The subject is a tetradecapeptide HEP-1 consisting of 14 consecutive amino acids, threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-valyl-glutamyl-arginyl-glutamyl-lysyl-glutamate (Figure 1), which are supposed to have immunomodulator and antiviral effects [30]. The mechanism of action may be based on altering the spectrum of synthesized interferons (IFNs) and cytokines [30]. A number of studies with limited patient sample sizes have reported positive effects of HEP-1 on the body’s immune response and overall medical state in a number of diseases, including decompensated caries, Hepatitis C, Human Immunodeficiency Virus (HIV), and Post-Acute Sequelae of Coronavirus disease (PASC) [30,31,32].

The molecule shows pronounced basic properties due to the presence of seven basic centers. High hydrophilicity results in low retention of HEP-1 when analyzed by RP HPLC, and the localization of the positive charge on the basic groups hinders fragmentation of the quasimolecular ion when detected by tandem mass spectrometry.

In order to study the pharmacokinetics of the drug for different routes of administration, it became necessary to develop a method that would be highly sensitive and selective for the determination of the peptide.

2.2. Mass Spectrum

Based on the calculated monomolecular mass of HEP-1, which is 1817.0 Da [33], we predicted the m/z values for single- and multicharged quasimolecular ions that can be detected in the mass spectrum of the total ion current during electrospray ionization in the positive mode (

Table 1. The calculated and obtained mass spectrum values of the human ezrin peptide-1 (HEP-1), which can be detected in scan mode.

n (H+)	m/z Calculated	m/z Obtained
1	1818.0	-
2	909.5	909.6
3	606.7	607.1
4	455.3	455.5
5	364.4	364.6

). The computed values matched the experimental values (Figure 2), but there were some deviations. These can be explained by the limited resolution of the detector. No single charged ions were detected due to the seven basic amino acid residues of HEP-1, which favor multicharged cations. According to Nadler et al. [34], the charge state of ionized peptides by electrospray increases with an additional basic amino acid independently of the type of basic residue. This can be beneficial because, according to Dongre et al. [19], multi-protonated peptides fragment more easily than the single-protonated forms of the same peptides, and a multicharged form decreases the number of degrees of freedom, which lowers the energy input into the fragment.

To establish the conditions for HEP-1 detection in the multiple reaction monitoring (MRM) mode, we analyzed the fragmentation mass spectra of three precursor ions (m/z: 607.1; 455.5, 909.6). However, we did not observe reliable fragmentation data regardless of the collision energy applied and the argon pressure inside the cell. It is known that lysine- and arginine-rich peptides at a given collision energy undergo fragmentation less easily than similar protonated peptides without a basic residue [19].

As a result, it proved challenging to identify the most suitable conditions for MS detection of HEP-1 in its native form due to the considerable energy required for the hydrophilic lysine- and arginine-containing peptide fragmentation. Furthermore, we observed that HEP-1 had a relatively low affinity for reversed-phase sorbents due to its high hydrophilicity and elution in “dead time” (retention time (RT) 0, 55 min). We considered using peptide derivatization to overcome these challenges.

2.3. Derivatizing Agent Selection

There are many basic amine groups in HEP-1, three lysine and three arginine residues, and no N-terminal modification for threonyl; therefore, we focused on finding amine derivatization agents. Furthermore, we considered that derivatization can reduce the gas-phase basicity of the peptide, which would reduce the energy required for fragmentation by collision-induced dissociation (CID) [19].

After a thorough literature review, we selected eight derivatizing agents of various chemical natures (sulfonyl chlorides, organic carbonates, and carboxylic acid anhydrides). These reagents are widely available and are used for the derivatization of amines, amino acids, and peptides, as well as for the protection of amino groups in organic synthesis:

• Dimethylaminonapthalene-5-sulfonyl chloride (dansyl chloride) [35];
• 4-Toluenesulfonyl chloride (tosyl chloride) [36];
• Phenylisothiocyanate (PITC) [37,38];
• N-(9H-fluorene-2-ylmethoxy)succinimide (Fmoc-OSu) [39,40,41];
• Di-tert-butyl dicarbonate (Boc anhydride) [42];
• N-(Benzyloxycarbonyloxy)succinimide (Cbz-OSu) [43]
• Benzoic anhydride [44]
• Propionic anhydride [39,45,46]

A brief description of the reaction conditions for each derivatizing agent and a step-by-step evaluation of success are presented in

Table 2. Derivatizing agents, reaction conditions and method development progress. Succeeded steps for each agent are marked as +, not succeeded as -.

Derivatizing Agent	Reaction Condition	Derivative Product Mass Spectra	4-Substituted RT	Informative Product Ion Scan	MRM Developed	Controlled Yield Reaction	Applied in Plasma	Achieved High Sensitivity
-	Native HEP-1 without derivatization	-	0.55	-	-	-	-	-
Dansyl chloride	50 °C for 30 min in a carbonate buffer (pH 9) with a mixed solvent (water-acetonitrile, 1:1)	+	4.63	-	-	-	-	-
Tosyl chloride		+	3.91	+	+	-	-	-
PITC		+	3.95	+	+	-	-	-
Fmoc-OSu	Room temperature in the dark for 30 min in triethylamine at pH 9	+	5.13	+	-	-	-	-
Boc anhydride		+	3.85	+	+	-	-	-
Cbz-OSu		+	4.08	+	+	-	-	-
Benzoic anhydride	Room temperature overnight	+	3.91	+	-	-	-	-
Propionic anhydride	50 °C for 30 min in a propionic acid solution	+	2.42	+	+	+	+	+

. For comparison, information on native HEP-1 without derivatization is provided.

All of the substances considered are capable of interacting with the amino groups of HEP-1 under certain conditions [38]. Garcia et al. postulated that at a pH less than 10, modification of the N-terminal amino group and the ε-amino groups of lysine residues is the most likely outcome, while the labeling of residues with a higher pK_a is possible at a pH more than 10 [46]. Direct labeling of an arginine side-chain is a much less probable outcome due to the high pK_a value of the guanidinium group (12.5). Methods for modifying the side-chain of arginine are known, but many of them are designed for arginine itself, and not for arginine in the peptide. Reactions often have low rates and yields and may require the addition of sodium or potassium hydroxide to deprotonate the guanidine group to increase its nucleophilicity [47]. We assume no reactions with arginine occurred in our study since the pH value during the derivatization was no more than nine, and we did not observe more than a quadruple of substituted derivatives: three substitutions for lysine residues, and one for N-terminus. Three arginine residues did not appear to be derivatized under the postulated conditions. Chromatographic analysis under conditions postulated in Table 3 and Table 4 revealed multiple peaks for peptide products with three or fewer substitutions, indicating the separation of isomers derivatized at different positions. All the mass spectra for peptide derivatives are presented in Supplementary Materials and in the main text for derivatives with propionic anhydride.

The interaction of HEP-1 with dansyl chloride, tosyl chloride, and a PITC reagent was conducted in a carbonate buffer at pH 9 in a mixed solvent (water-acetonitrile) with heating in an ultrasonic bath [36,38,48,49].

The use of dansyl chloride in the reaction mixture resulted in the detection of 3- and 4-substituted peptide derivatives, whose observed mass spectra matched with the theoretically calculated values (Table A1 and Table 2, Figure S1). However, as with the native peptide, the derivatized molecular ions had insufficient fragmentation to obtain informative mass spectra; therefore, we decided not to perform further derivatization with dansyl chloride.

Similar trends were observed when working with tosyl chloride (Table A1 and Table 2, Figure S2), although we observed minor fragmentation of quasimolecular ions for the tosylated derivatives. Furthermore, we developed MRM transitions (570.95 → 84.00; 570.95 → 91.00; 570.95 → 101.90; 570.95 → 154.90; 570.95 → 237.90; 760.95 → 83.90; 760.95 → 102.00; 760.95 → 154.90; 760.95 → 238.00). However, we did not find convenient conditions to control the yield of 3- and 4-tosylated derivatives. As a result, we found the process of tosylating HEP-1 to be inefficient and too challenging to control and continued the search for a superior derivatizing reagent.

The interaction of HEP-1 with PITC showed characteristics and complications analogous to those observed with tosyl chloride (Table A1 and Table 2, Figure S3). When the reaction was carried out for 30 min, only 1-, 2-, and 3-derivatized products of HEP-1 were detected. When we increased the reaction time to 1 h, we observed only 3- and 4-derivatized products. However, a further increase in time did not shift the product ratio toward the 4-derivatized form, for which we developed MRM transitions (787.15 → 94.15; 787.15 → 84.00). Due to an uncontrolled reaction yield, further investigation with PITC derivatives was considered inadvisable.

The interaction of HEP-1 with Fmoc-OSu, Boc anhydride, and Cbz-OSu was conducted in triethylamine (pH = 9) in the dark for 30 min. We identified the derivatives of Fmoc-OSu, Boc anhydride, and Cbz-OSu as 1-, 2-, 3-, and 4-substituted compounds (Table A1 and Table 2, Figures S4–S6). The 4-charged ions of the 4-substituted derivatives fragmented most efficiently, and we developed MRM for two of them (Boc anhydride: 740.30 → 57.05; 740.30 → 84.05; 740.30 → 129.00; 740.30 → 606.90; Cbz-Osu: 785.40 → 91.05; 1178.00 → 1504.40). However, the detector signal intensity was reasonable only at relatively high HEP-1 concentrations. As with the previously discussed reagents, we did not identify the optimal reaction conditions for the highest yield of the target 4-substituted derivatives.

Derivatization with benzoic anhydride was conducted at room temperature overnight. Among the reaction products, we found 2-, 3-, and 4-substituted peptide derivatives, but their quasimolecular ions did not appear to undergo fragmentation in the collision cell (Table A1 and Table 2, Figure S7).

In the experiment with propionic anhydride, HEP-1 predominantly yielded 4-substituted derivatives. We can control the ratio of reaction products easily by adjusting the amount of propionic anhydride added to the reaction mixture, the reaction temperature, and the reaction time. We did not detect derivatization products after conducting the reaction at room temperature, but it yielded 4-substituted derivatized peptides after 30 min in a 50 °C ultrasonic bath in acidic conditions. The mass spectrum for the substance corresponded with the calculated one (Table A1 and Table 2, Figure 3); moreover, we observed the fragmentation of multicharge ions of the 4-propionylated derivatives (681.30 → 73.95; 681.30 → 84.00; 681.30 → 101.90; 681.30 → 140.10), which allowed us to select and optimize detection conditions in the MRM mode. Consequently, we considered derivatization of HEP-1 with propionic anhydride to be the most efficient and continued further investigations.

The data on retention times (Table 2) showed that the lipophilicity of all the obtained 4-substituted derivatives is significantly higher than that of the native form of the peptide. The relative retention times of 3-substituted forms compared to 4-substituted ones had values in the range of approximately 0.8–0.9, and two to four peaks of isomeric forms of 3-substituted derivatives could often be detected on the chromatograms. We observed an even greater number of isomers for 2- and 1-substituted forms, if we could identify them among the reaction products. This phenomenon is due to the fact that derivatization occurs for four groups in the peptide structure, and with an incomplete reaction, various combinations of groups participating in the reaction are possible.

In the next phase of the research, we evaluated different methods for the preparation of blood plasma samples and selected optimal conditions for HEP-1 derivatization with propionic anhydride.

2.4. Sample Preparation Selection

Historically, three different sample purification methods have been used to prepare blood plasma samples. These include solid-phase extraction, liquid–liquid extraction, and plasma protein precipitation, as well as combinations and modifications of these methods [50,51]. In terms of complexity, protein precipitation is considered the simplest method, followed by liquid–liquid extraction, and then solid-phase extraction. In order to obtain a more economical technique, we paid particular attention to the protein precipitation method.

We studied the three basic methods of blood plasma protein precipitation using acetonitrile, methanol, and a 50% solution of trifluoroacetic acid (TFA) in water as precipitants. Simultaneously, we conducted propionylation of HEP-1 either directly in blood plasma or in the supernatant after protein precipitation. The schematics of the sample preparation procedures are presented in Scheme 1.

In addition, we performed parallel assays with blank blood plasma to confirm the specificity of the technique. For this purpose, we performed the same procedures sequentially with 200 µL of intact blood plasma, without the addition of HEP-1 and IS.

The experiment resulted in the detection of a 4-propionylated HEP-1 peak in all samples where derivatization was performed in blood plasma. We observed no peak corresponding to the test substance in the chromatograms of samples derivatized in the supernatant. This finding suggests that HEP-1 has a low recovery rate with acetonitrile and methanol. Consequently, we hypothesized that HEP-1 is co-precipitating with plasma proteins. For the optimized conditions of a sample preparation procedure, the recovery rate ranged from 50% to 62% across the tested concentration levels, as we established during method validation [52]. In the case of precipitation with TFA, there was no propionylation of HEP-1, likely due to the presence of a strong acid. Therefore, we assume derivatization in the supernatant after protein precipitation with TFA to be an inappropriate procedure.

Derivatization of HEP-1 in blood plasma using methanol as a precipitant yielded the strongest signal intensity. However, when we analyzed samples with different concentrations, we observed no linearity in the results obtained for the target 4-propionylated derivative. Therefore, we assumed a side reaction would occur and analyzed the sample in total ion current. The analysis revealed the presence of a substance with a molecular mass of approximately 2056 Da (m/z 514.9, 686.4, 1029.0). Mass spectrum is presented in Figure 4. The substance had a later retention time than the target compound (2.55 min for the byproduct and 2.42 min for the target product). We supposed that elevated temperatures within an acidic water-methanol solution would induce a side reaction with carboxylic groups of HEP-1, and the resulting product would be a methyl ester. Two mechanisms for the side reaction may be proposed: the formation of mixed anhydrides [53,54] and transesterification [55]. In the first case, propionic anhydride initially interacts with the carboxyl groups of the peptide, and a mixed anhydride is obtained, which is then destroyed by methanol with the formation of methyl esters. This hypothesis is strengthened by the observation that the mass spectrum showed correspondence with a calculated mass spectrum of the methylated derivative of 4-propionylated HEP-1.

We detected three poorly separated peaks on the chromatogram of the byproducts. It can be explained that esterification of carboxylic groups by methanol may occur on different side-chains of the HEP-1 [56]. Therefore, we directed future research to prevent the formation of methylated byproducts during derivatization and to increase the sensitivity of the technique by concentrating the sample. We decided to use methanol for precipitation but evaporate the supernatant with non-derivatized HEP-1 for further derivatization with an acetonitrile solution of propionic anhydride, which excludes methylated byproducts.

We found that HEP-1 recovery was approximately twice as high in methanol acidified with 4.76% propionic acid. We assumed that the acidic precipitant would result in improved extraction because the protein bonds would break down. After final optimization of sample preparation conditions, we fully validated the developed method [52]. An analogue of HEP-1 with valine replaced by leucine in the amino acid sequence was used as an internal standard. The linearity of the method was 5.00–1000.00 ng/mL. When studying the selectivity, matrix effect, and recovery rate, we found that the matrix composition (normal, hemolytic, or lipidemic blood plasma) did not significantly affect the results. The accuracy and precision of the method also met the acceptance criteria. It is important to note that the derivatized peptide was stable in the prepared samples at 2–8 °C for 24 h. Thus, the validation results confirmed that the developed method, including the stage of HEP-1 derivatization with propionic anhydride, can be used in clinical practice, for example, to study the pharmacokinetics of HEP-1.

3. Conclusions

In this study, we investigated the utility of derivatization techniques for the analysis of hydrophilic lysine- and arginine-containing tetradecapeptide HEP-1 in human plasma using RP HPLC-MS/MS. Due to the high hydrophilicity of HEP-1 and its poor fragmentation tendencies in MS/MS analysis, we examined various derivatizing agents to enhance both chromatographic separation and mass spectrometric detection.

Evaluating eight different derivatizing agents provided insight into their effectiveness in interacting with the reactive amino groups of the peptide: the N-terminal and three lysine ε-amino groups. Our goal was to obtain a quadruply substituted product with good MS/MS fragmentation and sensitivity. We did not expect the derivatization reaction with the arginine residues, and no more than quadruply derivatized HEP-1 was detected for any agent, which is consistent with theoretical considerations. Most agents, such as dansyl chloride, Fmoc-Osu, and benzoic anhydride, showed limited success due to the insufficient fragmentation of the derivatized HEP-1 products. More promising candidates, such as tosyl chloride, Boc anhydride, PITC, and Cbz-Osu, enabled the development of MRM transitions for their derivatized products. However, there were major complications associated with an uncontrolled yield of the target product. Among the evaluated agents, propionic anhydride demonstrated the best results. We determined the optimal conditions to obtain 4-propionylated HEP-1 efficiently with high reproducibility.

In addition to the derivatization process, we investigated methods for preparing human blood plasma samples. We effectively cleaned up the samples using a combination of traditional methods, such as protein precipitation and liquid–liquid extraction. This approach was simple and economical. Our results suggested that the native HEP-1 peptide co-precipitates with plasma proteins when traditional precipitants such as methanol and acetonitrile are used. However, recovery increased significantly when we precipitated plasma proteins with a propionic acid solution in methanol. This approach resulted in sufficient sensitivity to determine HEP-1 at a 5.00 ng/mL level without interference with components of blood plasma. Validation confirmed the applicability of the developed sample preparation scheme with derivatization by propionic anhydride for the analysis of HEP-1 in blood plasma samples. However, adaptation of the proposed method for the analysis of other peptides with similar properties (hydrophilicity and the presence of multiple basic groups) may require additional modifications.

In conclusion, our findings illustrate the role of derivatization in overcoming analytical challenges associated with hydrophilic peptides. Our research also demonstrates the potential of propionic anhydride as an effective agent for enhancing the detectability of HEP-1 and its potential suitability for other hydrophilic peptides containing lysine and arginine in analyses of complex biological samples. This method can be applied to similar peptides to enhance sensitivity and specificity in HPLC-MS/MS analysis.

4. Materials and Methods

4.1. Chemicals and Reagents

Reagents were used during investigation: acetonitrile (ACN) (LC-MS grade, Biosolve, Dieuze, France), formic acid (98%, PanReac, Barcelona, Spain), chloroform (99.9%, Honeywell, Ratingen, Germany), propionic acid (99.84%, BASF, Ludwigshafen, Germany), propionic anhydride (>99.8%, Sigma-Aldrich, Saint Louis, MO, USA), dimethylaminonapthalene-5-sulfonyl chloride (dansyl chloride, >98%, Sigma-Aldrich, USA), phenylisothiocyanate (PITC, 98%, Sigma-Aldrich, USA), 4-toluenesulfonyl chloride (tosyl chloride, >98%, Merck KGaA, Darmstadt, Germany), and benzoic anhydride (98%, Acros Organics, Geel, Belgium); methanol (MeOH, Chemically Pure), sodium bicarbonate (Reagent grade), and sodium carbonate (Reagent grade) were purchased from Chimmed Group, Moscow, Russia; N-(9H-fluorene-2-ylmethoxy)succinimide (Fmoc-Osu, >98), di-tert-butyl dicarbonate (Boc anhydride, >95%), N-(Benzyloxycarbonyloxy)succinimide (Cbz-Osu, >98%) and triethylamine (TEA, >99%) were obtained from TCI, Tokyo, Japan. Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Molsheim, France).

4.2. Apparatus and HPLC Conditions

Chromatographic separation and detection were performed using a Nexera high-performance liquid chromatograph with a tandem mass spectrometric detector LCMS-8040 (triple quadrupole, Shimazu, Tokyo, Japan), equipped with a gradient pump LC-30AD (Shimazu, Japan), column and sample thermostat CTO-20AC (Shimazu, Japan), degasserDGU-20A_5R (Shimazu, Japan), and automatic sampler SIL-30AC (Shimazu, Japan). Lab Solutions software (Ver. 5.97), Shimadzu Corporation, Japan, was used for primary data processing.

The chromatography method was performed using a Waters XBridge C18 Column, 5 µm, 4.6 mm × 50 mm. Gradient elution was performed using 0.1% formic acid in water (eluent A) and 0.1% formic acid in ACN (eluent B); the flow rate was 1.0 mL/min for 5 min for propionic anhydride derivatives and 7 min for others; gradients are presented in tables. An electospray ionization (ESI) interface was operated in positive mode, with the following set of operation parameters: capillary voltage, 5000 V; nebulizing gas flow, 5 L/min; desolvatation line temperature, 250 °C; heating block temperature, 400 °C; drying gas flow, 20 L/min; column temperature, 40 °C; injection volume, 2 μL.

Table 3. Mobile phase gradient for propionic anhydride derivatives.

Time, min	Eluent B, %
0.00–0.50	5
0.50–2.50	5 → 30
2.50–2.60	30 → 100
2.60–3.60	100
3.60–4.10	100 → 5
4.10–5.00	5

Table 3. Mobile phase gradient for propionic anhydride derivatives.

Time, min	Eluent B, %
0.00–0.50	5
0.50–2.50	5 → 30
2.50–2.60	30 → 100
2.60–3.60	100
3.60–4.10	100 → 5
4.10–5.00	5

Table 4. Mobile phase gradient for other derivatives.

Time, min	Eluent B, %
0.00–0.50	5
0.50–4.70	5 → 55
4.70–4.80	55 → 100
4.80–5.80	100
5.80–6.50	100 → 5
6.50–7.00	5

Table 4. Mobile phase gradient for other derivatives.

Time, min	Eluent B, %
0.00–0.50	5
0.50–4.70	5 → 55
4.70–4.80	55 → 100
4.80–5.80	100
5.80–6.50	100 → 5
6.50–7.00	5

4.3. Preparation of Solutions

4.3.1. Preparation of HEP-1 Stock Solution

The stock standard solution of HEP-1 was prepared in ACN/Water 1:1 in a concentration of 1 mg/mL by dissolving 2 mg of the drug substance in 2 mL ACN/Water 1:1 and stored at −20 °C, protected from light.

4.3.2. Preparation of Derivatizing Agents Solutions

Work solutions of derivatizing agents (dansyl, tosyl chloride, PITC, Fmoc-Osu, boc anhydride, Cbz-Osu, benzoic anhydride) were prepared at a concentration of 1 mg/mL by dissolving the calculated amount of the reagent in ACN.

4.3.3. Preparation of Buffer Solutions

A carbonate–bicarbonate buffer was prepared by mixing 850 mg of sodium carbonate with 840 mg of sodium bicarbonate, followed by ultrapure water added up to 100 mL (0.08 M sodium carbonate and 0.1 M sodium bicarbonate).

A work solution of TEA was prepared at a concentration of 1 mg/mL by dissolving the calculated amount of the reagent in ACN.

4.4. Preparation of Analytical Samples

4.4.1. Preparation of Dansyl, Tosyl, and PITC Derivative Samples of HEP-1

To 20 μL of HEP-1 stock solution 20 μL of carbonate-bicarbonate buffer was added, followed by the addition of 20 μL of a 1 mg/mL work solution of the derivatizing agent (dansyl, tosyl, or PITC) and 40 μL of water. Then, the solution was stirred in a vortex shaker for 10 s, followed by an ultrasonic bath at 50 °C temperature for 30 min (dansyl, tosyl) or 1 h (PITC). The mix was diluted by 300 μL of ACN, stirred, and transferred to a chromatographic vial.

4.4.2. Preparation of Fmoc-Osu, Boc Anhydride, Cbz-Osu Derivative Samples of HEP-1

In a dark environment, 20 μL of HEP-1 stock solution 20 μL of 1% TEA was added, followed by the addition of 20 μL of a 1 mg/mL work solution of the derivatizing agent (Fmoc-Osu, boc anhydride or Cbz-Osu) and 40 μL of water. Then, the solution was stirred in a vortex shaker for 10 s, followed by standing in a dark place at room temperature for 30 min. The mix was diluted by 300 μL of ACN, stirred, and transferred to chromatographic vials.

4.4.3. Preparation of Benzoic Anhydride Derivative Samples of HEP-1

To 20 μL of HEP-1 stock solution 20 μL of of 1 mg/mL work solution of the benzoic anhydride was added, followed by the addition of 60 μL of ACN. Then, the solution was stirred in a vortex shaker for 10 s, followed by night-standing at room temperature. The mix was diluted by 300 μL of ACN, stirred, and transferred to chromatographic vials.

4.4.4. Preparation of Propionic Anhydride Derivative Samples of HEP-1

To 20 μL of HEP-1 stock solution 5 μL of of 1% propionic anhydride in ACN was added, followed by the addition of 50 μL of water and 10 μL of propionic acid. Then, the solution was stirred in a vortex shaker for 10 s, followed by an ultrasonic bath at 50 °C for 30 min. The mix was diluted by 300 μL of ACN, stirred, and transferred to a chromatographic vial.

4.4.5. Preparation of Propionic Anhydride Derivative Samples of HEP-1 in Blood Plasma

A total of 190 μL of intact blood plasma was added to 10 μL of an ACN/Water 1:1 HEP-1 solution, followed by the stirring and adding of 420 μL of a 4.76% propionic acid solution in methanol, stirred in a vortex shaker for 10 s, followed by a 20 min standing period, and then centrifuged for 15 min at a relative centrifugal acceleration of 15,294× g. The supernatant was then transferred to 2 mL microcentrifuge tubes. In order to remove the methanol, 800 μL of chloroform was added to the supernatant. Following stirring and centrifugation, approximately 250 µL of the aqueous–methanol phase (upper layer) was obtained, with the majority of the methanol retained in the lower layer. The methanol–water layer was dried in a nitrogen current at room temperature, and then 60 µL of a 16.67% propionic acid solution in water and 55 µL of a 9.09% propionic anhydride solution in ACN were added. The derivatization reaction was conducted at 50 °C for 30 min. The excess propionic anhydride and acetonitrile were removed by evaporating the sample, after which the dry residue was redissolved in 100 μL of a 1:1 ACN-water mixture, stirred, and transferred to chromatographic vials.

In the Supplementary Materials, this sample preparation procedure is presented as a step-by-step protocol.