Author’s Accepted Manuscript Determination of sulfonamides in serum by on-line solid-phase extraction coupled to liquid chromatography with photoinduced fluorescence detection Natalia Arroyo-Manzanares, Francisco J. Lara, Diego Airado-Rodríguez, Laura Gámiz-Gracia, Ana M. García-Campaña

PII: DOI: Reference:

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S0039-9140(15)00165-4 http://dx.doi.org/10.1016/j.talanta.2015.03.012 TAL15445

To appear in: Talanta Received date: 19 December 2014 Revised date: 4 March 2015 Accepted date: 6 March 2015 Cite this article as: Natalia Arroyo-Manzanares, Francisco J. Lara, Diego AiradoRodríguez, Laura Gámiz-Gracia and Ana M. García-Campaña, Determination of sulfonamides in serum by on-line solid-phase extraction coupled to liquid chromatography with photoinduced fluorescence detection, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.03.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DETERMINATION OF SULFONAMIDES IN SERUM BY ON-LINE SOLIDPHASE EXTRACTION COUPLED TO LIQUID CHROMATOGRAPHY WITH PHOTOINDUCED FLUORESCENCE DETECTION Natalia Arroyo-Manzanares, Francisco J. Lara, Diego Airado-Rodríguez, Laura Gámiz-Gracia and Ana M. García-Campaña* Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain

Abstract An analytical method based on on-line solid-phase extraction coupled to liquid chromatography with photoinduced fluorescence detection has been developed to determine sulfonamides in serum. A home-made setup was used to percolate 3 mL of sample through a solid-phase extraction column. Analytes were retained onto the sorbent by an anion exchange mechanism which ensures an optimum compatibility with the subsequent chromatographic separation using a C-18 column and an on-line photoreactor in order to derivatize sulfonamides, which do not present native fluorescence. The method allowed the determination of 7 sulfonamides in serum samples previously deproteinized in less than 18 min and with limits of detection ranging between 1.8 and 3.6 mg/L. Relative recoveries between 91.5 and 102.1% were obtained with satisfactory precision since relative standard deviations were always below 10.5%.

Keywords: on-line solid-phase extraction, sulfonamides, liquid chromatography, photoinduced fluorescence, serum

Corresponding author: Ana M. García-Campaña ([email protected]) Phone: +34-958-242-385 Fax: +34-958-243-328

1. Introduction Sulfonamides are antibiotics that belong to the group of antibacterials. They inhibit a broad-spectrum of both gram positive and gram-negative bacteria, presenting chemotherapeutic activity against the infections caused by them as well as some protozoa. They are commonly prescribed in human and veterinary medicine against infection of digestive and respiratory tracts, affections of the skin, as well as for therapy of coccidiosis in animals [1,2]. In humans, for medical purposes, sulfonamides are normally administered orally through pharmaceutical preparations in a concentration range from 250 to 500 mg/L. Sulfonamides are well distributed in all body tissues. High concentrations can be found in bile, cerebrospinal fluid, prostatic fluid and sputum. They are metabolized in the liver but are primarily excreted unchanged in the urine. Taking into account their absorption rate, metabolism and elimination paths, typical concentrations in urine range between 0.001–1 µg/mL [3]. Regarding human blood, it is a common practice to maintain the blood levels of sulfonamides at approximately 0.1–100 µg/mL [3], however a significant variation in blood levels might be found when analyzing blood from different patients treated with the same doses. In general, levels ranging between 50 and 150 µg/mL are considered therapeutically effective for most infections and levels between 120 and 150 µg/mL are considered optimal for serious infections. Several reviews have been published including the different techniques and methodologies for sample treatment in the determination of sulfonamides in a great variety of matrices such as food, environmental samples, drugs or biological fluids [3, 4 , 5 , 6 ]. With regard to sample preparation, mainly for cleaning up and preconcentration, traditionally liquid-liquid extraction (LLE) and mainly solid phase extraction (SPE) have been two of the most commonly employed procedures [3], in spite of other modern microextraction techniques such as salting-out liquid–liquid extraction (SALLE) [31], liquid phase microextraction (LPME) [7], hollow fiber LPME [8], QuEChERS [9,10] or dispersive liquid–liquid microextraction (DLLME) [10,11]. In general, SPE continues to be the most applied sample preparation method in routine analysis [3,12] showing some advantages including the easy automation and on-line coupling with liquid chromatography (LC) which involves reduction of the exposure to hazardous solvents and higher sensitivity [13,14]. However, compatibility between the

elution step and the chromatographic separation must be taken into account. Solvents with high eluotropic strength are usually used to elute analytes from SPE cartridge but if analytes are injected in this solvent they would be poorly retained in the LC column, which would not be convenient for on-line SPE. In order to overcome this disadvantage an alternative is to use SPE sorbent based on ionic interactions. Analytes could be eluted just changing the pH and without the need of high percentages of organic solvents. If the pH required for eluting the analytes is similar to the pH of the mobile phase an optimum compatibility between SPE and LC will be ensured. Some of the anion exchange sorbents used were PRP-X100 poly(styrene–divinylbenzene) based trimethylammonium to determine glyphosate and aminomethyl phosphonic acid in water [15], Isolute SAX to determine insulin derivatives in biological matrices [16], methylcellulose-immobilized-strong

anion-exchanger

to

determine

aspirin

and

metabolites in plasma [17], Dionex IonPac AG16 guard column to determine iodide and thiocyanate in powdered milk and infant formula [18], weak anion exchange monolithic column to determine cefazolin sodium and cefotaxime sodium in human urine [19], Biomax AX to determine intracellular triphosphate metabolites in peripheral blood mononuclear cells [20,21], hypercrosslinked polymer resin (HXLPP) modified with 1,2ethylenediamine to determine a group of pharmaceuticals in water [22], Strata X-CW for therapeutic and abuse drugs in urine [ 23], Hysphere MM to determine folate catabolites in human biofluids [24] and Biobasic AX for domoic acid in shellfish [25]. Recently this strategy has been applied for the determination of quinolones in tap water and human urine using HyperSep Javelin Direct-Connect Retain-AX or HyperSep Uniguard Retain-AX without any sample pretreatment, only dilution of urine in water [26]. Reversed phase liquid chromatography (RP-LC) has been traditionally the most employed technique and in the last years ultra high-performance liquid chromatography (UHPLC) has increasingly been used for the rapid separation of these compounds, mainly with mass spectrometry (MS) detection [9,27,28,29,30]. Other detection systems coupled with LC comprise UV and DAD [11, 31 , 32 , 33 ], fluorescence previous derivatization with fluorescamine [10, 34 , 35 ], chemiluminescence [ 36 ] or electrochemical detection [37]. In relation to the application of on-line SPE coupling with LC for the determination of sulfonamides, some works have been reported using Oasis HLB for waters [38,39,40],

multiwalled carbon nanotubes in eggs and pork [41] or alumina for soils [42]. To the best of our knowledge, there is no report of the use of an anion exchange sorbent for their determination by on-line SPE coupled with LC. Although sulfonamides present poor native fluorescence, some methods have been proposed for their determination based on their native fluorescence or sensitized fluorescence [ 43 ]. Nevertheless, the use of different derivatization procedures to sensitize their fluorescent detection is a common practice. Chemical agents, mainly fluorescamine, have been typically employed for fluorescence labelling [10,34,35]. Enhanced fluorescence signals have been discovered and exploited for analytical methodology development, based in the formation of inclusion complexes of sulfonamides in cyclodextrins [44]. A special case of derivatization includes the use of light, which gives rise to photochemically-induced fluorescence (PIF) methods. The photochemical decomposition of sulfonamides has been reported [45,46,47] as well as the fluorescence properties of some important sulfonamides and their respective photoproducts in aqueous medium [ 48 ], concluding that UV irradiation induces enhanced fluorescence signals for heterocyclic sulfonamides meanwhile it induces decrease of fluorescence for non-heterocyclic ones. Sulfacetamide, sulfaguanidine and sulfametazine have been directly determined in milk and pharmaceutical preparations [49]. Other methodologies based on PIF for the monitoring of sulfonamides involved the use of first and second derivative techniques [50, 51, 52, 53], the coupling with flow injection analysis (FIA) [43, 54], the application of multivariate calibration strategies [55] or the proposal of a fluorimetric multioptosensor [56]. The aim of this work is to check the performance of the on-line coupling of SPE with LC using an anion exchange sorbent for the monitoring of sulfonamides in a biological matrix, such as serum, with minimum sample conditioning. Also, in order to increase sensitivity, PIF is proposed as detection system, using an on-line photochemical derivatization unit after the chromatographic separation. The proposed system could offer a high degree of automation and a satisfactory sensitivity combining on-line both SPE and photodegradation of analytes to obtain fluorescent products.

2. Materials and methods 2.1. Chemicals, reagents and samples All the reagents were analytical reagent grade, solvents were HPLC grade and sulfonamides were analytical standard grade (Vetranal, Sigma–Aldrich, St. Louis, MO, USA). Methanol (MeOH) and acetonitrile (MeCN) were supplied by Panreac (Madrid, Spain). Acetic acid and formic acid were obtained from Sigma-Aldrich (St Louis, MO, USA). A stock standard solution of 1000 µg/mL of each sulfonamide (sulfadiazine: SDZ; sulfapiridine: SPD; sulfamerazine: SMR; sulfamethazine: SMZ; sulfachloropiridazine: SCP; sulfamethoxazole: SMX; sulfadoxine: SDX) was prepared by dissolving 10 mg of the product in 10 mL of MeOH. The solutions were stable for at least 2 months, stored in the dark at 4 ºC. The working solutions were prepared by dissolving the appropriate volume in deionized water. Filters of 25 mm with 0.22 μm polyethersulfone membrane (Agela Technologies, DE, USA) were used for sample filtration. Ultrapure water (18.2 MΩ cm-1, Milli–Q Plus system, Millipore Bedford, MA, USA) was used throughout all the work. A serum pool from anonymous patients was obtained from a local hospital. 2.2. Instrumentation The chromatographic separation was carried out on a Jasco LC system consisting of a quaternary pump (PU-2089), an autosampler with 100 µL loop (AS-2055) coupled to a UV Derivatization module (LCTech, Dorfen, Germany) and subsequently to a fluorescence detector (Model FP 2020, Jasco). LC-Net II/ADC was used as the hardware interface between the LC system and the computer. Fluorescence chromatograms were acquired using ChromNav version 1.18.03. For on-line SPE experiments, the autosampler was removed and a setup consisting of a multisyringe pump with programmable speed (MultiBurette 4S, Crison Instruments, Alella, Barcelona, Spain), a low pressure selection valve (Rheodyne 5011, Supelco, Bellefonte, PA, USA) and a high pressure 6-port injection valve (Rheodyne 7725i, Supelco,

Bellefonte, PA, USA) were used instead. The SPE procedure was executed and controlled by software written in-house using Visual Basic 2010 (Microsoft, Redmond, WA, USA). The software was designed to control the position of commutation valves and the speed and direction of piston movement on the multisyringe pump. Figure 1 shows the on-line SPE – HPLC setup with the corresponding times for each step. 2.3. Chromatographic conditions LC separation was performed in a Luna C18 column (150×0.5 mm, 5 µm) from Phenomenex (Torrance, CA, USA). Mobile phase consisted of 5% acetic acid in water (solvent A) and 5% acetic acid in MeCN (solvent B) at a flow rate of 1.2 mL/min. The eluent gradient profile was as follows: 0 min: 5% B; 15 min: 25% B; 23 min: 60% B. Finally it was back to 5% B in 2 min and maintained for 5 min for column equilibration. The temperature of the column was 40 °C and the injection volume was 3 mL. Fluorescence detection was performed at their maximum excitation/emission wavelength 240/350 nm. Gain detector was set to 100. 2.4. On-line SPE procedure The SPE column was conditioned with 2 mL of MeOH containing 2% formic acid and 2 mL of H2O at a flow rate of 1 mL/min, and then 3 mL of sample was loaded at a flow rate of 0.5 mL/min. Before injection the sample solvent was removed from the column with 2 mL of H2O at a flow rate of 1 mL/min. Analytes were eluted in the first 5 minutes of the proposed gradient. Back-flush mode was used in order to reduce bandbroadening. Positions of both selection and injection valves were modified at the proper analysis time, as show in Figure 1, in order to synchronize SPE with the chromatographic separation. The SPE column was stored overnight in a mixture of H2O:MeCN (1:1, v/v). 2.5. Sample conditioning procedure 1.00 mL of MeOH:MeCN 1:1 (v:v) was added to 0.500 mL of a human serum pool for protein precipitation. The mixture was vortexed during 30 s and further centrifuged at 9000 rpm for 10 min at 20 ºC. Later, 0.250 mL of the supernatant were diluted up to 250.0 mL with ultrapure water and further analyzed by the proposed SPE-LC-PIF methodology.`

3. Results and discussion 3.1. On-line solid-phase extraction optimization The conditions for the on-line SPE procedure in order to monitor sulfonamides were based on a previous paper developed in our group [26], testing also four different anion exchange SPE columns: a home-made one (3 cm x 2.1 mm) packed with Oasis MAX sorbent (30 µm particles) and three commercially available columns such as HyperSep column Retain-AX (2 cm x 2.1 mm), HyperSep Uniguard Retain-AX (1 cm x 2.1 mm) and HyperSep Javelin Direct-Connect (1 cm x 2.1 mm). The anion exchange moiety in these four columns consisted of a polymer partially functionalized with quaternary amine groups. A Strata-X 25 µm (2 cm x 2.0 mm) column from Phenomenex (Torrance, CA, USA), which is based on non-polar interactions, was also tested. As we expected, the non-polar column gave very broad peaks and poor sensitivity was obtained. Also, the HyperSep column Retain-AX gave the higher back pressure (35 bar), and the multisyringe pump was not able to deliver the required volume; therefore it was discarded. The rest of the columns had no problem: HyperSep Uniguard Retain-AX (5 bar), HyperSep Javelin Direct-Connect (16 bar) and the home-made one with Oasis MAX sorbent (6 bar). The latter was discarded due to a slightly higher irreproducibility most likely due to manual packing. No significant differences were observed between HyperSep Uniguard Retain-AX and HyperSep Javelin Direct-Connect. Actually they use the same sorbent and have the same dimensions (1 cm x 2.1 mm). Both of them were used in this paper and they lasted at least one month. According to the previous method, the SPE column was conditioned with 2 mL of MeOH containing 2% formic acid and 2 mL of H2O at a flow rate of 1 mL/min. Different volumes of diluted sample were tested, achieving the best results using 3 mL of sample loaded at a flow rate of 0.5 mL/min. A linear relationship between peak area and injection volume was observed, showing the proportionality of the method and the high retention capacity of the sorbent. Before injection the sample solvent was removed from the column with 2 mL of H2O at a flow rate of 1 mL/min. Further washing stages were not considered since interfering compounds at the retention times of the analytes of interest were not detected under the optimized conditions. Analytes were eluted in the first 5 minutes of the proposed gradient.

3.2. Chromatographic separation It is known that the solvent has an important effect on the development of PIF from sulfonamides [57]. PIF signals are significantly favored in alcoholic media, in terms of sensitivity, rapidness and precision. In this case, the effect of the solvent was studied by injecting standard solutions of the studied sulfonamides by separate in a flow injection system coupled with the same photoreactor and detector, using different solvents and solvent mixtures. MeOH, MeCN and different aqueous-organic mixtures were assayed, trying to mimic typical mobile phases in RP-LC. The best results, in terms of photoreaction yield, were obtained in the presence of MeCN. For this reason, the mobile phase selected was water and MeCN as solvents A and B, respectively. Then, the percentage of acetic acid in mobile phase was optimized between 0 and 10%. A concentration of 5% acetic acid in both, solvents A and B, gave the highest signals and good peak shape. The gradient was studied in order to get the best separation, peak shape and sensitivity in the shortest time. The following gradient was selected: 0 min: 5% B; 15 min: 25% B; 23 min: 60% B. Finally it was back to 5% B in 2 min. Although the run time for each injection was 30 min (including the cleaning and conditioning of the column), under optimum conditions all analytes were eluted in about 16 min. The effect of column temperature was examined in the range of 25–45 °C; an optimum value of 40 °C was chosen, as a compromise between analysis time and column life. The flow rate was also tested between 0.9–1.3 mL/min, selecting a final optimum value of 1.2 mL/min, as higher flow rates involved higher pressure without a significant improvement in the analysis. 3.3. Characterization of the method In order to check the suitability of the method for the determination of seven sulfonamides in serum, an exhaustive characterization was carried out. So, linear dynamic ranges, limits of detection (LOD) and quantification (LOQ), precision and trueness were evaluated.

3.2.1. Calibration curves and performance characteristics Calibration curves were obtained using different serum samples spiked at five concentrations of sulfonamides in the range of 30 to 125 mg/L. Each concentration level was prepared in duplicate, submitted to the subsequent protein precipitation process and dilution 1:1000 and then, the extract was injected in duplicate. The statistical parameters were calculated by least-square regression, and LODs and LOQs were considered as 3×S/N ratio and 10×S/N ratio, respectively. Table 1 summarizes the results. Satisfactory determination coefficients confirm that sulfonamide analytical responses were linear over the studied ranges. Moreover, with the low LOQs obtained, the seven sulfonamides can be determined at concentrations normally found in this kind of matrix. It is worth to mention the high dilution factor applied, which reduces drastically the interferences while increasing selectivity. Combined with the on-line preconcentration process and the high sensitivity offers by the PIF detection, this on line SPE-PIF method is a very simple alternative to control sulfonamides in this complex biological matrix. 3.2.2. Precision study The precision of the whole method was evaluated in terms of repeatability (intraday precision) and intermediate precision (interday precision). Repeatability was assessed by application of the whole procedure on the same day to three samples (experimental replicates) spiked at three different concentration levels of each sulfonamides. Each sample was injected in triplicate (instrumental replicates). Intermediate precision was evaluated with a similar procedure, spiking and analyzing five samples in different days, injected in triplicate. The results, expressed as %RSD of peak areas are shown in Table 2, showing values lower than 10.5%.

3.2.3. Recovery studies In order to check the trueness of the proposed method, recovery experiments were carried out on serum samples. These samples were spiked at three different

concentration levels, processed as described previously and injected in triplicate into the on line SPE-HPLC-PIF system. Recoveries were calculated as (signal of a spiked sample / signal of a spiked extract) x 100. The results are shown in Table 3 and as it can be seen, satisfactory values were obtained.

4. Conclusions The use of SPE coupled on-line with LC has been demonstrated as a useful alternative for the monitoring of sulfonamides in serum samples. An anion exchange sorbent facilitates the on-line coupling, since a high content of organic solvent when the separation method starts is avoided making compatible the SPE elution and chromatographic separation. Just with the right pH in the mobile phase is possible to achieve a complete elution of the retained analytes. Also, the combination of this strategy with the PIF detection by using an on-line photochemical derivatization unit reduces sample manipulation in the production of fluorescence derivatives from sulfonamides, instead of using more complex chemical derivation procedures, increasing sensitivity, selectivity and sample throughput. It is important to mention that the high sensitivity and selectivity obtained permits a simple sample treatment based mainly on dilution, which allows the injection of the diluted sample directly in the online SPE setup.

Acknowledgements The Andalusia Government (Junta de Andalucía) supported this work (Project Ref: P12-AGR-4268). Natalia Arroyo Manzanares thanks the “Junta de Andalucía” for a predoctoral grant. Diego Airado-Rodríguez thanks the Spanish Ministry of Science and Innovation for a “Juan de la Cierva” contract.

Table 1. Statistical and performance characteristics of the proposed method in human serum samples Standard Error of

Analyte Slope

Slope

Intercept

Standard

Linear

Error of

range

Intercept

r2

LOD

LOQ

(mg/L) (mg/L)

(mg/L) -7012

SDZ 4881

6626

125

81 -16792

SPD 4979

7680

94 43151

11836

2540

SMZ

17654

98631

SCP

12250

154 33499

27943

343

8.1 – 125

8678

SDX 9119

8.8 – 125

SMX 6985

8.4 – 125

217

2300

6.0 – 125

145

6694

9.7 – 125

SMR 9371

11.9 –

348

28346

7.6 – 125

0.997

3.6

11.9

0.996

2.9

9.7

0.997

1.8

6.0

0.988

2.5

8.4

0.957

2.6

8.8

0.972

2.4

8.1

0.983

2.3

7.6

Table 2. Precision study in serum samples (% RSD of peak areas) SDZ

SPD

Level 1 Level 2 Level 3

6.7 5.4 5.7

7.2 6.0 5.8

Level 1 Level 2 Level 3

8.1 8.8 8.5

7.9 7.5 8.4

SMR SMZ SCP SMX Repeatability (n = 9) 7.5 5.3 6.5 5.9 4.8 4.2 5.7 6.2 5.7 5.0 5.1 5.4 Intermediate precision (n = 15) 9.0 9.9 8.5 7.6 10.5 9.0 8.4 8.3 8.7 8.1 7.5 7.1

Level 1: 30 mg/L; Level 2: 50 mg/L; Level 3: 125 mg/L.

SDX 6.0 6.3 7.1 8.8 7.6 7.9

Table 3. Recovery (%) study in serum samples (%RSD of peak areas is given in parentheses)

n=9

Level 1

Level 2

Level 3

SDZ

92.3 (6.7)

94.7 (5.4)

93.6 (5.7)

SPD

91.5 (7.2)

93.8 (6.0)

95.7 (5.8)

SMR

100.9 (7.5)

99.6 (4.8)

99.9 (5.7)

SMZ

95.3 (5.3)

96.4 (4.2)

94.9 (5.0)

SCP

98.7 (6.5)

102.1 (5.7)

99.0 (5.1)

SMX

92.3 (5.9)

93.6 (6.2)

96.4 (5.4)

SDX

91.7 (6.0)

92.6 (6.3)

91.5 (7.1)

Level 1: 30 mg/L; Level 2: 50 mg/L; Level 3: 125 mg/L.

Figure 1. On-line SPE–LC setup diagram. G: gradient, Sc: Solid-phase extraction column, Hv: High pressure 6-port valve, Sv: Low pressure selection valve. The table shows the position sequence for the different valves, the duration of each step and how they are synchronized with the chromatographic separation.

Figure 2. On-line SPE-LC-PIF chromatogram of a human serum sample spiked with 7 sulfonamides at 30 mg/L (a) and the corresponding blank (b). (1: SDZ; 2: SPD; 3: SMR; 4: SMZ; 5: SCD; 6: SMX; 7: SMD).

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post-column

chemiluminescence determination of sulfonamide residues in milk at low concentration

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using as

bis[4-nitro-2-(3,6,9-

chemiluminescence

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combined

with

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binary

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of

fluorescence

for

sulfonamides

in

pharmaceuticals, milk and urine, Anal. Chim. Acta 600 (2007) 164-171. [ 57 ] M. Sánchez Peña, F. Salinas, M.C. Mahedero, J.J. Aaron, Analysis of sulfamethazine in the presence of sulfamerazine or sulfadiazine by first-derivative photochemically induced fluorescence, Talanta 41 (1994) 233-236.

Highlights On-line SPE coupled to LC with photoinduced fluorescence detection to monitoring sulfonamides – The anion exchange sorbent for SPE makes highly compatible the online coupling with LC – Analysis of 7 sulfonamides in serum previously deproteinized in less than 18 min - Relative recoveries between 91.5 and 102.1% with RSD lower than 10.5 %.

Figure 1

Time (min) 0.0

MeOH-FA (1 mL/min)

18.0

Loading

5.0

Separation

Injection

HV Position

HPLC

Idle

SV Position

Syringe Pump

A B A: 5% acetic acid in H2O B: 5% acetic acid in MeCN

G SC

20.0

Detector

22.0

25.0

28.0

30.0

H2O (1 mL/min)

Equilibration

Sample (0.5 mL/min)

Sample

Waste

Column

H2O (1 mL/min)

MeOH H2O + 2% FA

SV

HV

Pump

Figure 2

Figure 2

Injection

HV Position

Time (min) 0.0

Syringe Pump

SC

20.0

Detector

22.0

25.0

28.0

30.0

H2O (1 mL/min)

Equilibration

Sample (0.5 mL/min)

Sample

Waste

Column

H2O (1 mL/min)

MeOH H2O + 2% FA

SV

HV

Pump

MeOH-FA (1 mL/min)

18.0

Loading

5.0

Separation

Idle

HPLC

G

A B A: 5% acetic acid in H2O B: 5% acetic acid in MeCN

SV Position

*Graphical Abstract (for review)