The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.
A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.
Sarcopenia is a progressive skeletal disorder related to an accelerated loss of muscle in the elderly population. This condition increases the risk of falls and leads to limited activities of daily living. Sarcopenia is present in about 5%-10% of persons over 65 years old and about 50% of persons aged 80 years or older1. No specific drugs have been approved for the treatment of sarcopenia, so prevention with physical activity and a well-balanced diet is important1,2. Nutritional interventions with specially formulated foods enriched with dairy protein and essential amino acids have shown positive results in preventing sarcopenia2. In other studies, authors have included vitamins and antioxidants, like vitamin E and isoflavones, in the diet, increasing the benefits for muscle gain on the waist and hips3.
Brosimum alicastrum Sw. (Ramón) is a tree that grows in the Mexican tropical regions; it has been consumed by Mayan cultures due to its high nutritional value4. It is a good source of protein, fiber, minerals, and phenolic antioxidants, such as chlorogenic acid5. Since it can be ground into powder and used in baking products or consumed in beverages, recent studies have evaluated the incorporation of Ramón seed flour (RSF) into different foods to improve their nutritional value. An RSF-supplemented cappuccino-flavored beverage was formulated, which was high in dietary fiber and had more than 6 g of protein per serving, and was highly accepted by consumers; thus, it was considered a potential alternative for meeting special dietary requirements6. In a follow-up study, RSF was also used to formulate a muffin and a new beverage rich in protein, dietary fiber, micronutrients, and phenolic antioxidants. The muffin and beverage were used in a dietary intervention for elderly individuals, who consumed both products twice per day for 30 days. After this period, the nutritional and sarcopenic status of the participants improved, and the total phenolic content of plasma increased7. However, the determination of total phenolic compounds in plasma was carried out by a spectrophotometric method, so identification of the actual phenolic compounds that were absorbed was not possible; moreover, this method is not completely specific for phenolic compounds, so some overestimation may occur8.
Identification and quantification of the phenolic compounds that are absorbed after consumption of foods rich in these antioxidants is a difficult task but is necessary to demonstrate the biological activity of these phytochemicals. The bioavailability of most phenolic compounds is low; less than 5% of them can be found without structural transformation in plasma. Phenolic compounds undergo several biotransformations, such as methylation, sulfonation, or glucuronidation, which are carried out by enterocytes and hepatocytes9. Phenolic compounds are also biotransformed by the microbiota into bacterial catabolites that may exert their beneficial effects in the body after being absorbed into the plasma10. For example, phenylacetic acid is a product of the bacterial transformation of flavonoids and oligomeric proanthocyanidins, which can inhibit up to 40% of bacteria (Escherichia coli) adhesion in the urinary tract after cranberry consumption11.
The structural diversity of naturally occurring phenolic compounds, added to the diversity of their metabolites and their low bioavailability, makes their identification in plasma even more challenging. Metabolomic profiling, using spectroscopic analysis platforms like nuclear magnetic resonance (NMR) and tandem mass spectroscopy (MS/MS), is probably the best approach to achieve this goal; unfortunately, the equipment is not easily accessible, and the development of analysis protocols is still limited12. Several studies have reported MS/MS coupled with a separation system (such as liquid chromatography) as a strategy for reducing the complexity of mass spectra in metabolomic studies. The recent introduction of ultra-high-performance liquid chromatography (UPLC) separation methods has reduced the time of analysis and increased the resolution and sensitivity compared with conventional high-performance liquid protocols, so UPLC-MS/MS systems have rapidly been widely accepted by the analytical metabolomics community13. In this way, some studies have investigated phenolic metabolites and detected glucuronidated derivatives from caffeic acid, quercetin, and ferulic acid, as well as sulfonated derivatives from syringic and vanillic acid in the plasma of individuals after cranberry intake14. Previous protocols have intended to find phenolic compounds and phenolic metabolites in biofluids such as plasma. These protocols were based on identification and quantification by high-performance liquid chromatography (HPLC) coupled to a UV-vis detector15. Nevertheless, such protocols require the use of authentic standards to assess absolute identification and accurate quantification. A wide range of studies have identified the most common metabolites in biofluids (sulphonated, glucuronidated, and methylated forms) by UPLC-MS and UPLC-MS/MS; however, a large part of the bacterial metabolites has not been reported due to the lack of databases that contain their complete information16. Metabolite identification is complicated by the cost and commercial availability of metabolite standards. Therefore, the best strategy may be untargeted or semi-targeted MS/MS metabolite analysis, which relies on the use of molecular feature information (m/z, monoisotopic exact mass, isotopic distribution, and fragmentation pattern) to determine the chemical identity and compares it with freely available online databases that contain polyphenol metabolites identified in biofluids after the consumption of polypolyphenol-richts12. The most important databases used in UPLC-MS/MS studies for the identification of phenolic compounds and their metabolites are the Human Metabolome Database (HMDB), LipidBlast Library, METLIN Library, and other complementary databases, such as PubChem, ChemSpider, and Phenol Explorer17.
In the present study, a semi-targeted UPLC-MS/MS method was developed to analyze the plasma samples of the group of elderly persons involved in the RSF-containing muffin and beverage consumption study7. Data from different free online databases of plasma metabolites were collected and integrated into a specialized database. This database can be accessed automatically by the equipment software to identify the polyphenolic metabolites in the five plasma samples before and after the 30-day nutritional intervention. This is done to identify the main phenolic compounds, or their metabolites, that are absorbed from the specially formulated functional foods designed for the prevention of sarcopenia.
The plasma samples used in this protocol were collected in a previous study following all the ethical guidelines and approved by the Institutional Ethics and Bioethics Committee (CIEB-2018-1-37) from the Universidad Autónoma de Ciudad Juárez. The complete protocol for the extraction and identification of the phenolic compounds and metabolites in plasma by UPLC-MS/MS is represented in Figure 1.
Figure 1: Schematic representation of the extraction and identification of phenolic compounds and metabolites in plasma by the semi-targeted UPLC-MS/MS method. Please click here to view a larger version of this figure.
1. Sample preparation
2. UPLC-MS/MS analysis
Time (min) | Solvent A (0.1 % formic acid in HPLC water) | Solvent B (100 % acetonitrile) |
0 to 1 | 90 | 10 |
1 to 4 | 70 | 30 |
4 to 6 | 62 | 38 |
6 to 8 | 40 | 60 |
8 to 8.5 | 40 | 60 |
8.5 to 9 | 90 | 10 |
Table 1: Mobile phase gradient used for the separation of phenolic compounds by UPLC.
Ionization mode | Negative |
Drying gas | Nitrogen at 340 °C, flow rate 13 L/min |
Nebulizer pressure | 60 psi |
Capillary voltage | 175 V |
MS scan masses | 100-1100 m/z |
MS/MS scan masses | 50-1000 m/z |
Table 2: Ionization parameters for the MS/MS analysis.
3. Database construction
4. Data analysis
The step-by-step process for the identification of phenolic metabolites through the semi-targeted UPLC-MS/MS analysis, in negative mode, of plasma samples is depicted in Figure 2. First, the total ion chromatogram (TIC) from the plasma phenolics extract (obtained after protein precipitation of the total plasma sample) was obtained through the instrument's qualitative software. Then, the extracted ion chromatogram was used, and the exact mass and fragmentation pattern (MS/MS analysis) of each signal (or molecular feature) were compared with those of a specific personal database created also in the instrument's software. Signals with a mass match of less than 5 ppm were assigned a molecular formula from the database. Finally, the isotopic distribution of each signal was compared to that of the assigned molecular formula to achieve the final tentative identification. Compounds that a) were identified only in one replicate or b) presented an area lower than 10,000 were treated as false identifications. From this analysis, a total of 25 phenolic compounds and metabolites were identified in the plasma samples (Table 3). In this list, both phenolic compounds and their metabolites, such as 3-hidroxyphenylvaleric acid and isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, were found. Since the negative ionization mode is best suited for all classes of phenolic compounds, except anthocyanins, these compounds could not be detected with the present method. If anthocyanins are important components of the food matrix, the positive mode should be also used.
Phenolic metabolites | R.T. (min) | Formula | Precursor | Experimental mass | Theorical mass | Difference (ppm) | |
2,3-Dihydroxybenzoic acid | 0.622 | C7H6O4 | 153.0203 | 154.0273 | 154.0266 | 4.2 | |
2-Hydroxyhippuric acid | 8.631 | C18H33NO4 | 410.1648 | 411.1725 | 411.1717 | 1.8 | |
3,4-Dihydroxytoluene | 2.239 | C7H8O2 | 123.0451 | 124.0524 | 124.0524 | -0.25 | |
3-Hydroxyphenylvaleric acid | 6.717 | C11H14O3 | 193.0874 | 194.0947 | 194.0943 | 2.12 | |
5-(3',4'-dihydroxyphenyl)-valeric acid | 4.293 | C11H14O4 | 209.0823 | 210.0894 | 210.0892 | 0.68 | |
6-Hydroxyenterodiol | 9.201 | C18H22O5 | 317.1387 | 318.1465 | 318.1467 | -0.65 | |
Ajugol | 3.889 | C15H24O9 | 347.134 | 348.1418 | 348.142 | -0.59 | |
Benzoic acid | 3.915 | C7H6O2 | 121.0296 | 122.0367 | 122.0368 | -0.28 | |
Carnosic acid | 6.785 | C20H28O4 | 331.1905 | 332.1979 | 332.1988 | -2.58 | |
Carnosol | 6.347 | C20H26O4 | 329.1764 | 330.1842 | 330.1831 | 3.43 | |
Catechol | 0.892 | C6H6O2 | 109.0297 | 110.037 | 110.0368 | 1.91 | |
Glycitin | 6.01 | C22H22O10 | 445.1155 | 446.1228 | 446.1213 | 3.4 | |
Hesperetin | 6.01 | C16H14O6 | 301.0718 | 302.0796 | 302.079 | -1.81 | |
Hippuric acid | 1.396 | C9H9NO3 | 178.051 | 179.058 | 179.0582 | -1.16 | |
Homovanillic acid | 0.823 | C9H10O4 | 181.0503 | 182.0576 | 182.0579 | -1.88 | |
Isopropyl 3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoate | 6.177 | C12H16O5 | 239.0926 | 240.0999 | 240.0998 | 0.48 | |
Phenylacetic acid | 5.666 | C8H8O2 | 135.0444 | 136.0518 | 136.0524 | -4.92 | |
Phloretic acid | 2.811 | C9H10O3 | 165.0556 | 166.0626 | 166.063 | -2.41 | |
Protocatechuic aldehyde | 1.094 | C7H6O3 | 137.0247 | 138.0311 | 138.0317 | -4.5 | |
Secoisolariciresinol | 8.837 | C20H26O6 | 361.1656 | 362.1729 | 362.1729 | -0.23 | |
Vanillin | 2.508 | C8H8O3 | 151.04 | 152.0471 | 152.0473 | -1.82 | |
Epicatechin 3'-O-glucuronide | 9.342 | C21H22O12 | 465.1024 | 466.109 | 466.1111 | -4.64 | |
Gomisin M2 | 5.234 | C22H26O6 | 385.1676 | 386.1746 | 386.1729 | 4.38 | |
Irisolidone | 6.145 | C17H14O6 | 313.0727 | 314.0798 | 314.079 | 2.33 | |
Urolithin C | 6.753 | C13H8O5 | 243.0294 | 244.0368 | 244.0372 | -1.69 |
Table 3: Tentative identification of phenolic compounds and metabolites in plasma samples by the semi-targeted UPLC-MS/MS method.
To evaluate the effectiveness of the designed method for the identification of the main phenolic compounds, or their metabolites, that were absorbed from the RSF-containing muffin and beverage, five aleatory samples of the study participants, obtained before and after the 30-day intervention, were analyzed. The relative abundance of each compound was calculated by dividing the area under the curve (AUC) after treatment by the AUC before treatment. From this analysis, it was possible to observe that some compounds only appeared in the samples obtained before the treatment, others remained unchanged, while some of them increased after consumption of the functional foods. Table 4 shows the list of 12 phenolic compounds that showed an increase in plasma after the 30-day consumption of the RSF-containing foods. Phenylacetic acid was the only metabolite found consistently in higher concentrations after the treatment. Glycitin, a glycosylated isoflavone, and 3-hydroxyphenylvaleric acid (a phenolic metabolite) increased in three of the five samples but decreased in the other two. Gomisin M2, a lignan, was detected in three of the five samples only after the nutritional intervention. The other phenolic compounds (such as hesperetin, secoisolariciresinol, and vanillin) and metabolites (such as 2-hydroxyhippuric acid) were found only in one sample and only after the treatment.
Sample (AUC after treatmet/AUC before treatment) | |||||||
Compound | Formula | 1 | 2 | 3 | 4 | 5 | |
2-Hydroxyhippuric acid | C18H33NO4 | T | nd | nd | nd | nd | |
3-Hydroxyphenylvaleric acid | C11H14O3 | 1.30 | 2.69 | 2.69 | 0.62 | 0.62 | |
6-Hydroxyenterodiol | C18H22O5 | nd | nd | T | nd | nd | |
Glycitin | C22H22O10 | 1.88 | 1.07 | 1.07 | 0.43 | 0.45 | |
Hesperetin | C16H14O6 | T | nd | nd | nd | nd | |
Phenylacetic acid | C8H8O2 | 4.06 | T | T | T | 1.28 | |
Phloretic acid | C9H10O3 | T | nd | nd | nd | nd | |
Protocatechuic aldehyde | C7H6O3 | T | nd | nd | nd | nd | |
Secoisolariciresinol | C20H26O6 | T | nd | nd | nd | nd | |
Vanillin | C8H8O3 | T | nd | nd | nd | nd | |
Gomisin M2 | C22H26O6 | nd | T | T | T | nd |
Table 4: List of phenolic compounds that increased in the plasma of elderly individuals after 30-day consumption of RSF-containing foods. Data are the ratios of the abundance (AUC) of each compound after treatment compared to their abundance before the treatment. T indicates that the compound was only identified in the sample after the treatment. Nd: not detected.
Figure 2: Protocol for the identification of phenolic compound metabolites by semi-targeted UPLC-MS/MS. Please click here to view a larger version of this figure.
The identification and quantification of the bioactive phytochemicals that are absorbed after consumption of a food or food supplement are crucial for demonstrating and understanding the health benefits of these compounds and the foods containing them. In the present work, the UPLC-MS/MS method was developed, aimed only at the identification of the main phenolic compounds and their metabolites that increased in concentration in plasma after a 30-day nutritional intervention with two food products specially formulated for the elderly. It was assumed that, if one compound increased or appeared in plasma only after the intervention period, that compound had been absorbed and/or biotransformed from the foods used in the intervention.
UPLC-MS/MS is one of the preferred technologies for metabolomic studies in which many low molecular weight compounds are analyzed simultaneously in complex samples to assess the effect of some treatment or environmental alteration13. This can be done through targeted and non-targeted (or global) methods. Targeted analyses focus on a known, small number of metabolites of interest. They are the best option for quantifying compounds with available standards, providing the best specificity and sensitivity; however, these methods must be thoroughly optimized for the selected metabolites and are incapable of detecting unexpected compounds in the samples12. In other studies, HPLC coupled with a UV-vis detector has been used to identify phenolic acids and metabolites in plasma, but this type of study has used analytical standards to identify and quantify the compound of interest15. In another study, the addition of an internal standard was proposed18; however, only ferulic and caffeic acids and their metabolites were analyzed in this work. On the other hand, the synthesis of internal standards of phenolic metabolites has been proposed as an alternative to the lack of commercial analytical standards19. Nevertheless, the synthesis of a large variety of phenolic compounds is difficult, time-consuming, and expensive. Non-targeted methods attempt to detect and identify as many molecular compounds as possible and are primarily hypothesis-generating12. They can identify several hundreds of compounds in one sample by assigning formulas to detectable chromatographic signals with characteristic features, such as m/z or measured accurate mass, chromatographic retention time, isotopic fingerprint, etc., so the data generated by these analyses are very abundant and can be difficult to interpret20. Since the objective of the present method was not to obtain a full metabolic profile of the plasma samples (which would be an untargeted approach) but to identify the major phenolic compounds and phenolic metabolites (previously unknown) in them, a semi-targeted approach was designed. For this, all the identified signals were extracted automatically with the instrument’s software and then compared with a self-created database that contained 645 phenolic compounds and their metabolites, which were obtained from free online databases. The database included reference MS/MS fragmentation patterns of the compounds that were used for the assignment of the compound name and formula, which allows for more accurate identification of compounds in the sample12.
The most critical steps in the protocol were 1) sample pretreatment (extraction and concentration of phenolic compounds and metabolites from plasma samples); 2) the construction of a complete and specific database for the semi-targeted analysis of the samples and identification of all the possible compounds belonging to a specific compound class; and 3) the selection of the parameters used by the instrument’s qualitative software (mass match tolerance ppm, and score%) to accurately identify the compounds in the sample. In sample pretreatment, caution must be taken to avoid loss of the compounds of interest, recover them at a high final concentration, and be able to eliminate interfering compounds. In the creation of the specific database, it is critical to conduct a thorough literature search and then use free online databases to obtain specific chemical data that will be used for compound identification in the sample. The selection of the best values for mass match and score required previous validation of the analytical method using standards and known samples21,22.
Once the protocol was implemented, the common problems and their recommended solutions were the following: 1) No compounds were identified in the samples. This was due to the low concentration of the compounds and could be solved by drying the sample and re-dissolving it in a smaller volume. 2) Signals appeared in the TIC, but compounds were not identified. This could be due to a failure in mass calibration, so the difference between experimental and theoretical masses was high. In this case, the instrument’s mass calibration should be performed according to the manufacturer and model of the equipment. A second reason could be an incomplete personal database, so it is critical to constantly check and maintain the database. Masses of compounds suspected to be of interest can be checked and compared with freely available online databases or new scientific literature. 3) Signals appeared in blank runs. This indicates contamination and can be solved by cleaning the autosampler needle and column. The standard cleaning protocol is to perform some runs using methanol, then isopropanol, and finally acetonitrile as the mobile phase. Then the column is re-equilibrated, running the mobile phase for at least 30 min.
The major difficulties in the development of this kind of method are 1) the low bioavailability, in general terms, of all phytochemicals, including phenolic compounds, which translates to very low plasma concentrations16; 2) the high diversity of phenolic compounds present in foods, which is increased by their metabolism and biotransformation that occur in human enterocytes and hepatocytes and the colonic microbiota23; 3) the lack of standards (some standards exist but are very difficult to obtain) for many compounds, especially for the metabolites, and the existence of some unknown or uncharacterized compounds23; and 4) variable responses among individuals, both in absorption and metabolism of phytochemicals14. To overcome the problem of low plasma concentrations of phenolic metabolites, they should be extracted and concentrated. This can be achieved by micro solid-phase extraction14 or, as in the present work, by the addition of solvents that precipitate proteins and dissolve phenolic compounds and metabolites, followed by solvent evaporation and resuspension in a smaller volume19. This technique is simple, economic, and adequate for a small number of samples. However, the recovery of compounds of interest could be easily improved by using larger plasma volumes, so the final concentration of all the compounds would be higher. The high diversity of phenolic metabolites and lack of standards is the reason why a semi targeted approach was used to identify the compounds increased by the intervention before attempting to quantify them. By using a specially created database instead of a completely untargeted analysis, it was possible to ignore the numerous primary metabolites in plasma, focusing only on the phenolic compounds and their metabolites. An important limitation of this database, specific for phenolic compounds and their human metabolites, was the lack of mass spectral information for many compounds and their metabolites, which reduces the accuracy of the compound identification.
Large variability among individuals is regularly observed in studies evaluating absorption and metabolism of phenolic compounds, both quantitively and qualitatively. In the present results, one individual presented 18 compounds in the plasma sample taken after the intervention, while the others showed only 9 or 10. Moreover, many phenolic compounds and metabolites were found only in the baseline samples (before the intervention), and they also varied among individuals. Overall, more than half of the identified compounds showed higher concentrations (AUC) before than after the intervention or were found only in the pre-intervention samples. Therefore, it was necessary to compare individual samples taken before and after the intervention to understand which compounds were actually increased by it. The only metabolite that consistently increased after the treatment was phenylacetic acid, a microbial catabolite of flavan-3-ols24 that has been found in the plasma of individuals in acute studies of the consumption of phenolic-rich foods14,25. Most of the studies that have identified and quantified phenolic metabolites in plasma were acute studies, in which the compound concentrations were monitored during the 24 h after consumption of the phenolic-rich meal, observing that, after 24 h, their concentrations were near the basal value19,25. Therefore, it is understandable that the samples analyzed in the present work showed low numbers and concentrations of phenolic metabolites. Recently, Zhang et al.25 evaluated the consumption of red raspberry, both in acute studies and after 4 weeks of supplementation. They observed that both acute and chronic interventions increased phenolic compounds and their metabolites, while after the 4-week supplementation, the basal plasma concentration of some metabolites increased (urolithins, cinnamic, phenylpropionic, and hippuric acids) and of others decreased (conjugated anthocyanin derivatives).
A final examination of the method developed in the present paper shows that, in most parts, it was well suited for the objective for which it was created. The sample pretreatment was simple and effective, and the UPLC separation and semi-targeted MS/MS identification of phenolic metabolites were achieved. This semi-targeted method can be useful as a first screening approach to identify phenolic compounds that can be absorbed from different food matrixes, as well as the phenolic metabolites present in human plasma. Moreover, the protocol is useful because it allows identification when no standards from phenolic compounds or metabolites are available. Finally, the method uses a small plasma quantity, which is critical in most in vivo studies where plasma samples are scarce. However, it is recommended that, for future studies, an acute study should be carried out before the chronic intervention to better identify the main phenolic compounds and metabolites absorbed from the specially formulated foods.
The authors have nothing to disclose.
The authors are grateful for the financial support from CONACYT, Mexico (CB- 2016-01-286449), and UACJ-PIVA (Projects 313-17-16 and 335-18-13). OAMB wishes to thank CONACYT for his Ph.D. scholarship. Technical support from the Multimedia Production office from UACJ is gratefully acknowledged.
Acetonitrile | Tedia | Al1129-001 | LC Mass spectrometry |
Autosampler | Agilent Technologies | G4226A | 1290 Infinity series |
C18 reverse phase column | Agilent Technologies | 959757-902 | Zorbax Eclipse plus C18 2.1×50 mm, 1.8 μm; Rapid resolution HD |
Centrifuge | Eppendorf | 5452000018 | Mini Spin; Rotor F-45-12-11 |
Column compartment with thermostat | Agilent Technologies | G1316C | 1290 Infinity series |
Diode Array Detector (UV-Vis) | Agilent Technologies | G4212B | 1260 Infinity series |
Electrospray ionnization source | Agilent Technologies | G3251B | Dual sprayer ESI source |
Formic acid | J.T. Baker | 0128-02 | Baker reagent, ACS |
Mass Hunter Data Acquisition | Agilent Technologies | G3338AA | |
Mass Hunter Personal Compound Datbase and Library Manager | Agilent Technologies | G3338AA | |
Mass Hunter Qualitative Analysis | Agilent Technologies | G3338AA | |
Microcentrifuge tube | Brand | BR780546 | Microcentrifuge tube, 2 mL with lid |
Pure ethanol | Sigma-Aldrich | E7023-1L | 200 proof, for molecular biology |
Q-TOF LC/MS | Agilent Technologies | G6530B | 6530 Accurate Mass |
Quaternary pump | Agilent Technologies | G4204A | 1290 Infinity series |
Syringe filter | Thermo Scientific | 44514-NN | 17 mm, 0.45 μm, nylon membrane |
Thermostat | Agilent Technologies | G1330B | 1290 Infinity series |
Vial | Agilent Technologies | 8010-0199 | Amber, PFTE red silicone 2 mL with screw top and blue caps |
Vial insert | Agilent Technologies | 5183-2089 | Vial insert 200 μL for 2mL standard opening, conical |
Water | Tedia | WL2212-001 | LC Mass spectrometry |