Automated Purification and Analytical Reinjectionof a Small Molecule Drug, Probenecid, on a Gilson
LC/MS Dual Function System
Application Note PHA0413
Keywords
High Pressure Liquid Chromatography (HPLC), Mass Spectrometry (MS), preparative
HPLC/MS, analytical HPLC/MS, fraction collection, fraction reinjection, probenecid,TRILUTION® LC, conditional logic fraction collection

Introduction
Combined UV and mass‐based detection and fraction collection following
preparative liquid chromatography (LC/MS) represents a well‐established method forpurifying target compounds. Typically these systems utilize both UV and MS
detectors, employing conditional logic “on the fly” to determine whether collection
criteria set by the user is met based on UV signals and MS selected ion signals. This
technique offers the benefits of enhanced selectivity (exclusion of unwanted
fractions and attendant post‐collection processing) and real time as well as post‐collection mass spectral confirmation data, thus overcoming the inherent limitations
associated with purifying target compounds with HPLC using conventional UV
detection alone.
To provide additional confirmation data on the collected fractions of interest, bothpreparative‐ and analytical‐scale LC/MS can be conducted. The two methods(preparative collection and analytical) are performed on partially separate liquidchannels configured using a common liquid handling platform with dual injection
modules to enable full automation of the purification step and subsequent analyticalreinjection step. This configuration eliminates the need for manual intervention at
any point in the purification and analytical processes.
This application note describes the functionality of a Gilson LC/MS system for boththe purification and identification of probenecid, a well‐known uricosuric drug
commonly used to treat gout.1 The effects of the make‐up solvent on the generationand optimization of mass spectra for this compound are also evaluated.
Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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Materials & Methods
LC/MS System Setup and Conditions
Sample Preparation
The test sample was prepared using 5 mg/mL each of ephedrine, 4‐acetaminophenol,
chlorpheniramine, verapamil, and probenecid dissolved in a 60:30:10 solution of water,
methanol, and dimethyl sulfoxide (DMSO).
Gilson LC/MS System Components
 GX‐281 Liquid Handling Platform with Platinum Z Injection Module (5 mL loop) Analytical Direct Injection Module 155 UV/VIS Detector; .01 sensitivity; 0.2 mm flow cell
 MRA Splitter set to 1:1000 split ratio (19) FLEXAR SQ 300 Single Quadrupole MS Detector with four SIM (selective ionmonitoring) channels
 307 Make‐Up Pump
Components of the dual function system include a preparative‐scale column andinjector for purification and an analytical‐scale column and injector for sample
verification. Using TRILUTION LC 2.1 SP5 software, conditional logic fraction collection
was employed to collect probenecid, while a selective reinjection function allowed forautomated reinjection of the collected fraction for verification on the analyticalsystem.

Preparative LC/MS Conditions


HPLCColumn: Phenomenex Luna 21x50 mm
Mobile Phase: A: 0.1% formic acid in water B: 0.1% formic acid in methanol
Gradient: 10‐90% B in 7 min
Flow rate: 20 mL/min






MS
ESI mode: Positive
Make‐up solvent: 0.1% formic acid in
MeOH (0.3 mL/min flow rate)
Scan range: 100‐500 amu
Scan rate: 5 scans/sec

Probenecid Fraction Collection and Reinjection onto Analytical System
The fraction containing probenecid was collected using a conditional logic method inTRILUTION® LC. Conditional logic (AND) was applied to two channels: 1) primary – UV
(254 nm) slope criteria; 2) secondary – target mass criteria, i.e., probenecid SIM (m/z285.1; level 1,500,000).

Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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Analytical LC/MS Conditions



HPLC
Column: Phenomenex C18 6x50 mm
Mobile Phase: A: 0.1% formic acid in water B: 0.1% formic acid in methanol
Gradient: 10‐90% B in 2 min
Flow rate: 2 mL/min
MS
ESI mode: Positive
Make‐up solvent: 0.1% formic acid in
MeOH (0.3 mL/min flow rate)
Scan range: 100‐500 amu
Scan rate: 5 scans/sec

Results
The test sample was first processed on the preparative LC/MS system, with theprobenecid fraction (RT: 7.512 min and m/z 285.1) collected using conditional logic on
two channels (UV wavelength and target mass) (Figures 1 and 2). As indicated inFigure 2, the protonated probenecid ion ([M+H]+=286.30) was assigned an abundanceof 81 relative to the base peak at m/z 340.30. The base peak was suspected to be theresult of a methanol cluster on the sodium adduct (m/z 308.30).

Probenecid

Figure 1. Chromatogram showing UV‐based fractionation and detection ofcompounds in the test sample. Gradient: 10‐90% organic mobile phase(0.1% HCOOH in MeOH) in 7 min.

Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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Sodium adduct +methanol cluster
ProtonatedadductSodiumadduct

Figure 2. Probenecid fraction collection
spectrum using a 0.1% formic
acid in methanol make‐up
solvent with a flow rate of 0.3mL/min. The relative
abundance of the protonatedprobenecid ion([M+H]+=286.30) is indicated in
the red box.

Under TRILUTION® LC control, the collected fraction was then automatically reinjectedfrom the fraction tube onto the analytical system for further compound verification(Figures 3 and 4). Similar to the mass spectrum shown in Figure 2, the base peak was thesuspected methanol cluster on the sodium adduct at m/z 340.20. The relative abundanceof the probenecid protonated adduct (48) was notably low compared to that of theidentical adduct in the spectrum given by the original collected fraction.

Probenecid

Figure 3. Probenecid fraction reinjection on an analytical system. Gradient: 10‐90%
organic mobile phase (0.1% HCOOH in MeOH) in 2 min.

Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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Sodium
adduct +methanolcluster

SodiumadductFigure 4. Probenecid fraction
reinjection spectrum
using the make‐up
Protonatedsolvent and flow rateadductspecified above (Figure2). Red box indicatesthe protonatedprobenecid adduct.

As indicated above, the base peaks in the probenecid fraction collection and reinjection
spectra (Figures 2 and 4) were assumed to have originated from a methanol cluster on
the sodium adduct ion when the make‐up solvent consisted of formic acid in methanol.
To eliminate this adduct from the spectra, the experiment was repeated using a make‐up solvent consisting of formic acid in water. Use of the alternative solvent yielded anearly identical chromatogram (Figure 5), yet the mass spectra showed considerable
differences between the two make‐up solvents with regard to compound ionization.
The peak corresponding to the methanol cluster on the sodium adduct (m/z 340.20) wasabsent from the spectra produced when an aqueous make‐up solvent was used (Figure6). As such, the protonated adduct was designated as the base peak with a relative
abundance of 100.

Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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Probenecid

Figure 5. Fraction collection when a 0.1% formic acid in water make‐up solvent wasused with a flow rate of 0.3 mL/min. Gradient: 10‐90% organic mobilephase (0.1% HCOOH in MeOH) in 7 min.

Protonated adduct(base peak)Figure 6. Probenecid fraction reinjection spectra when a 0.1% formic acid in watermake‐up solvent was used with a flow rate of 0.3 mL/min. The peakcorresponding to the protonated probenecid adduct had the highest
abundance (100; indicated in red boxes) and was therefore designated as thebase peak.
Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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Configuration of a standard LC/MS purification system with a second injection valve
allows for integration of an analytical system with a preparative system, thereby
reducing manual intervention during the reinjection step. Here, an important
pharmaceutical compound, probenecid, was successfully purified from a sample
mixture containing several other compounds. Based on both UV and mass spectra,
the probenecid fraction was collected and reinjected onto an analytical system forabsolute verification. Use of a second injector allowed the transition frompreparative to analytical LC/MS to be entirely automated. Thus, the Gilson dual
function LC/MS system with automated fraction collection and reinjection
represents a systematic means of purifying and verifying fractions containing
pharmaceutical and other compounds of interest in the laboratory, allowing
laboratory chemists to focus on other activities and to avoid error‐prone manualsteps.

References
1. Silverman, W., Locovei, S., Dahl, G. Probenecid, a gout remedy, inhibits
pannexin 1 channels. Am. J. Physiol. Cell Physiol. (2008) 295: C761‐C767.

Gilson, Inc.
Middleton, WI 53562 USA
Telephone: 800‐445‐7661

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