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Journal of Chromatography A, 1339 (2014) 174–184

Contents lists available at ScienceDirect

Journal of Chromatography A

jo ur nal ho me pag e: www.elsev ier .com/ locate /chroma

oupling state-of-the-art supercritical fluid chromatography andass spectrometry: From hyphenation interface optimization to

igh-sensitivity analysis of pharmaceutical compounds

lexandre Grand-Guillaume Perrenoud, Jean-Luc Veuthey, Davy Guillarme ∗

chool of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland

r t i c l e i n f o

rticle history:eceived 25 November 2013eceived in revised form 26 February 2014ccepted 1 March 2014vailable online 11 March 2014

eywords:FC–MSHPSFC–MS

nterfacing approachetection sensitivityharmaceutical application

a b s t r a c t

The recent market release of a new generation of supercritical fluid chromatography (SFC) instrumentscompatible with state-of-the-art columns packed with sub-2 �m particles (UHPSFC) has contributedto the reemergence of interest in this technology at the analytical scale. However, to ensure perfor-mance competitiveness of this technique with modern analytical standards, a robust hyphenation ofUHPSFC to mass spectrometry (MS) is mandatory. UHPSFC–MS hyphenation interface should be able tomanage the compressibility of the SFC mobile phase and to preserve as much as possible the chromato-graphic separation integrity. Although several interfaces can be envisioned, each will have noticeableeffects on chromatographic fidelity, flexibility and user-friendliness. In the present study, various inter-face configurations were evaluated in terms of their impact on chromatographic efficiency and MSdetection sensitivity. An interface including a splitter and a make-up solvent inlet was found to bethe best compromise and exhibited good detection sensitivity while maintaining more than 75% of thechromatographic efficiency. This interface was also the most versatile in terms of applicable analyti-cal conditions. In addition, an accurate model of the fluidics behavior of this interface was created fora better understanding of the influence of chromatographic settings on its mode of operation. In thesecond part, the most influential experimental factors affecting MS detection sensitivity were identi-fied and optimized using a design-of-experiment approach. The application of low capillary voltage and

high desolvation temperature and drying gas flow rate were required for optimal ESI ionization andnebulization processes. The detection sensitivity achieved using the maximized UHPSFC–ESI-MS/MSconditions for a mixture of basic pharmaceutical compounds showed 4- to 10-fold improvementsin peak intensity compared to the best performance achieved by UHPLC–ESI-MS/MS with the sameMS detector.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Supercritical fluid chromatography (SFC) is currently experi-ncing a remarkable rebirth of interest in the field of separationcience. The recent reconsideration of this technique has beenrimarily triggered by the shortage of acetonitrile in 2008, which

ncreased the operating costs of liquid chromatography (LC). Inhis context, SFC has been considered as a greener alternative1,2]. There are also many other advantages offered by SFC that

ave been recently rediscovered by a wider audience. From aheoretical point of view, supercritical conditions allow significantncreases in analytes diffusion (DM), while the low mobile phase

∗ Corresponding author. Tel.: +41 22 379 34 63; fax: +41 22 379 68 08.E-mail address: davy.guillarme@unige.ch (D. Guillarme).

ttp://dx.doi.org/10.1016/j.chroma.2014.03.006021-9673/© 2014 Elsevier B.V. All rights reserved.

viscosity maintains a reasonable column pressure [3]. Thus, bothfast analysis at high linear velocity and enhanced chromatographicresolution with long columns can be easily achieved with SFC.Originally more dedicated to the analysis of lipophilic compounds,modern SFC has also been applied as a powerful technique for theanalysis of molecules exhibiting a broad range of polarity includingpharmaceuticals [4,5], natural products [6], ionic compounds [7],and even more recently peptides [8] thanks in part to the modifi-cation of the supercritical CO2 mobile phase with small amount ofpolar organic solvents [9]. Using such a binary mobile phase, thetechnique is still referenced as supercritical fluid chromatographyfor the sake of simplification even if, above a certain amount of

organic modifier, the mobile phase is generally no longer rigorouslyunder supercritical state but rather in a subcritical state. Neverthe-less, no phase disruption between these two states is observed onthe detected signal and the advantageous mobile phase properties

. / J. Ch

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A. Grand-Guillaume Perrenoud et al

re conserved in the subcritical region. Practically, SFC offers thedditional advantage that it can be easily used in reversed phasend normal phase without the need for any major change in termsf analytical conditions. Nowadays, a huge effort is consented inhe development of polar stationary phase for SFC that can pro-ides alternative selectivity compared to reversed phase LC (RPLC).he current interest for the technique is particularly marked athe analytical scale, since a new generation of SFC instrumentsdapted to the current analytical standards entered the market in011. Modern analytical SFC systems benefit from technologicaldvances in the pumping system and backpressure regulationllowing better control of the compressibility of the mobile phase10]. In addition, these new systems integrate the technical fea-ures of ultra-high performance LC (UHPLC), namely higher upperressure limits and reduced void volumes [11], allowing betterompatibility with the most recent stationary-phase technologiesuch as a sub-3 �m core–shell and fully porous sub-2 �m particles12–15]. With the advent of state-of-the-art columns, the kineticerformance achieved with new SFC setups is comparable to thosebserved in UHPLC [16,17], suggesting the introduction of theerm ultra-high performance SFC (UHPSFC) [6,16].

Due to its high sensitivity and selectivity, mass spectrometrys considered as one of the most powerful and versatile detec-ion methods. LC–MS platforms are currently considered the goldtandard for most analytical applications including investigationsf complex mixtures and biological matrices [18,19]. The potentialf SFC–MS has not been as thoroughly realized but is nonethe-ess promising and holds many advantages, particularly in termsf alternative selectivity and complementarity to LC–MS [20–22].FC–MS hyphenation was first described in the 1980s [23]. At thisime, chemical ionization (CI) or electron impact (EI) ionizationources were employed in capillary SFC [24]. With the develop-ent of packed-column SFC and use of a non-negligible proportion

f organic modifier in the mobile phase, SFC–MS quickly adoptedhe use of LC-like atmospheric pressure ionization (API) sources.ighly versatile and robust atmospheric pressure chemical ioniza-

ion (APCI) [25] and electrospray ionization (ESI) [26] sources wereoth successfully used in the early 1990s and are still considered asrst choice when using SFC–MS systems [27–29]. Due to the physi-al nature and compressibility of SFC mobile phase, moving columnffluent toward the ionization source is less straightforward than inC. Routing interfaces should limit the risk of analyte precipitationaused by SFC mobile phase loss of solvating power and densityrop related to its decompression. Such effects can be disastrousor chromatographic fidelity, peak shapes and detector response30]. Thus, dedicated interfaces including the control of backpres-ure (passive or active restrictor) must be employed. An extensiveeview focusing on the advantages and drawbacks of SFC–MS inter-aces available in 2005 was published by Pinkston [30]. Most ofhese interfaces could still be mounted on the last generation ofFC instrumentations and commercial ones have been proposed.owever, no study assessing their compatibility with UHPSFC–MS

pecifications, their flexibility in terms of operating conditions andheir ability to maintain the high chromatographic performanceave been published.

The present study investigates the influence of different inter-ace geometries and configurations with or without the use of a

ake-up fluid on the chromatographic performance and on theS detection sensitivity. A complete characterization of the most

romising interface was also conducted to evaluate the influence ofhromatographic conditions such as flow rate, mobile phase com-osition and backpressure on the analyte quantity or concentration

hat reaches the API probe. Finally, the absolute detection sensitiv-ty achieved using representative but maximized UHPSFC–MS/MSonditions were compared to that observed with separately fullyptimized UHPLC–MS/MS approach for a mixture of basic drugs.

romatogr. A 1339 (2014) 174–184 175

2. Experimental

2.1. Reagents and columns

Pressurized liquid CO2, 3.0 grade, (99.9%) was purchasedfrom PanGas (Dagmerstellen, Switzerland). LC–MS grade sol-vents (methanol, ethanol, isopropanol, acetonitrile and heptane)were purchased from VWR (Radnor, PA, USA). Formic acidand ammonium hydroxide (ULC–MS grade) were purchasedfrom Biosolve BV (Valkenswaard, The Netherlands). Water wasobtained from a Milli-Q Water Purification System from Millipore(Bedford, MA, USA). Alprazolam, clonazepam, prazepam, triazo-lam and methadone were purchased from Lipomed AG (Arlesheim,Switzerland). Hydroxyzine, indapamide, noscapine, papaverine,theophylline and polyethylene glycol (PEG) were purchasedfrom Sigma–Aldrich (Buchs, Switzerland). Acquity UPC2 BEH(100 mm × 3.0 mm, 1.7 �m) and Acquity UPLC BEH C18 columns(50 mm × 2.1 mm, 1.7 �m) for UHPSFC and UHPLC experiments,respectively, were purchased from Waters (Milford, MA, USA). Dif-ferent column dimensions were chosen for both chromatographictechniques in order to limit the instrumental contribution to bandbroadening in both cases.

2.2. Instrumentation

2.2.1. UHPSFC systemThe Waters Acquity UPC2 system was equipped with a binary

solvent delivery pump, an autosampler that included a 10 �L loopfor partial loop injection, a column oven, a UV detector fitted withan 8 �L flow-cell and a two-steps (passive + active) backpressureregulator. The passive component maintains pressure higher than104 bar while the active component allows further back pressureincrease and fine backpressure adjustments. The connection tubebetween the injector and column inlet was 600 mm long (pre-heater included) and had an I.D. of 0.175 mm; the capillary locatedbetween the column and detector was 600 mm long and had anI.D. of 0.175 mm. On this instrument, the extra-column volume ofthe system was measured at 59 �L and the extra-column variancewas measured at 85 �L2, whereas the gradient delay volume was440 �L. The hyphenation interface and splitter for UHPSFC–MS aredetailed in Section 2.2.4.

2.2.2. UHPLC systemThe Waters Acquity UPLC was equipped with a binary solvent

manager, an autosampler with a 2 �L loop operating in the full-loop injection mode, and a column oven. The connection tubebetween the injector and column inlet was 300 mm long (preheaterincluded) and had an I.D. of 0.125 mm. The column outlet capillarywas 250 mm long and had an I.D. of 0.125 mm and was directlyconnected to the ESI probe. On this instrument, the extra-columnvolume of the system was measured at 13 �L and the extra-columnvariance was measured at 8 �L2, while the gradient delay volumewas 100 �L.

2.2.3. MS/MS detectorBoth chromatographic systems were hyphenated with the same

Waters TQD triple quadrupole mass spectrometer fitted with aZ-spray electrospray ionization (ESI) source. Ionization and MSdetection were carried out in the ESI positive mode and withselected reaction monitoring (SRM), respectively. The source tem-perature, cone gas flow and source extractor voltage were identical

in both UHPSFC and UHPLC modes (120 ◦C, 20 L/h and +3 V, respec-tively). The capillary voltage, desolvation gas temperature andflow rate were optimized using a design-of-experiments (DoE)approach.

176 A. Grand-Guillaume Perrenoud et al. / J. Ch

FiB

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2

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2

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ig. 1. Schematic representations of the three UHPSFC–ESI-MS/MS interfaces testedn this study. (A) Pre-BPR splitter with sheath pump interface and (B) Pre-UV andPR splitter without sheath pump interface.

Cone voltages, collision energies and SRM transitions were opti-ized for each compound by direct infusion. The optimal valuesere not affected by the chromatographic mode and remained

qual in UHPSFC and UHPLC. Finally, dwell times and inter-channelelays were set to 15 and 5 ms, respectively, to achieve a sufficientumber of data points across the peaks [31].

.2.4. UHPSFC–MS splitter interface configurationsThe UHPSFC system was hyphenated with an MS detector

sing 2 different interface configurations illustrated in Fig. 1 andescribed as follows: (A) pre-UV-BPR-split: The UHPSFC columnutlet tube was replaced by a 400 mm long, 0.175 mm I.D. cap-llary connected to a zero-dead-volume T-union allowing mobilehase splitting. The first part of the flow was directed toward theS detector using a 750 mm long, 0.050 mm I.D. PEEK-sil transfer

ine, while the other part was directed toward the back-pressureegulator (BPR) through the UV detector using the original 600 mmong, 0.175 mm I.D. tube; and (B) pre-BPR-split + make-up pump:his interface kit purchased from Waters was composed of twoerial zero-dead-volume T-unions connected to the UV detector orolumn outlet with 410 mm long, 0.175 mm I.D. tubing. CO2 mis-ible make-up liquid delivered by a Waters HPLC 515 make-upump was added and mixed to the chromatographic effluent in thepstream T-union, while the downstream T-union acted as a flowplitter. A fraction of the total flow was directed from the down-tream T-union to the ESI source through a 750 mm long, 0.050 mm.D. PEEK-sil transfer line, while the remaining mobile phase wasirected to the BPR via a 1270 mm long, 0.250 mm I.D. connection.

.2.5. SoftwareInstrument control, data acquisition and data handling of

he UHPSFC–MS and UHPLC–MS systems were performed withasslynx 4.1 (Waters). The physicochemical properties of the

harmaceutical compounds were predicted using ACD/ADMEuite software (version 5, Advanced Chemistry Development, Inc.,oronto, ON, Canada, www.acdlabs.com, 2012). Calculations andodeling of splitter behavior were performed using MS Excel Soft-are. Modde software (version 7.0.0.1, Umetrics, Umeå, Sweden)as used for DoE generation, statistical data processing and

esponse-surface modeling.

.3. Procedure and methodology

.3.1. UHPSFC–MS interface evaluationsThe influence of the splitter interface on chromatographic

nd detection performance was assessed for the 2 configurations

escribed in Section 2.2.4 using a mixture of 6 pharmaceuti-al compounds (100 ppb each) dissolved in a ternary injectionolvent system consisting of EtOH:IPA:heptane, 1:2:7, v:v:v. TheHPSFC mobile phase was an isocratic mixture of 92/8 (v/v)

romatogr. A 1339 (2014) 174–184

CO2/MeOH + 20 mM NH4OH delivered at 1 mL/min for the totalflow introduction interface and at 2.0 mL/min for both the pre-UV-BPR-split and pre-BPR-split + make-up pump interfaces. The 20 mMNH4OH adjunction was required to achieve reasonable peak shapefor basic compounds under SFC conditions [32]. Column tempera-ture was set at 40 ◦C and BPR at 120 bar for the two latter interfaceconfigurations. For the pre-BPR-split + make-up pump interface,pure MeOH was used as the sheath liquid at a fixed flow rate of0.6 mL/min. The capillary voltage, desolvation gas temperature andflow rate were set at 1.0 kV, 400 ◦C and 800 L/h, respectively. Themost intense SRM transitions were monitored for each compound,e.g., prazepam (325 → 271), theophylline (181 → 124), noscap-ine (414 → 220), clonazepam (316 → 270), papaverine (340 → 202)and indapamide (366 → 132). Detection intensity was obtainedfrom individual extracted ion current (XIC). The effects of addi-tional splitter tubing on peak broadening, selectivity and generatedpressure were further investigated for the pre-BPR-split + make-uppump interface using the same analytical conditions and comparedto the chromatographic performance and behavior achieved forthe UHPSFC–UV configuration. The same mixture was used whenUV detection was performed at 210 nm, but the concentration wasincreased to 10 ppm.

2.3.2. Splitter behavior modelingThe total amount of MeOH entering the ESI source, split ratio

and sample dilution factor of the pre-BPR-split + make-up pumpinterface were modeled based on splitter tubing lengths and inter-nal diameters. For this purpose, the Hagen–Poiseuille relationshipwas employed:

F = �d4

128�× �P

L(1)

where F is the MeOH flow rate entering the MS, d is the capil-lary internal diameter, � is the fluid viscosity, �P is the changein capillary pressure and L is the capillary length. The viscosity ofthe final fluid (homogenous phase composed of CO2 and MeOHcoming from both pumps) was estimated based on experimentalcorrelations proposed by Ouyang [33] and applied recently to SFCby Grand-Guillaume Perrenoud et al. [11].

To check the reliability of predictions, the MeOH flow ratedelivered through the transfer capillary toward the ESI probe wascollected for 3 min using a homemade MeOH trap. For this pur-pose, 81 experimental conditions including 3 UHPSFC flow rates(1.0, 2.0 and 3.0 mL/min), 3 UHPSFC mobile phase compositions(5, 10 and 20% of MeOH in CO2 (v/v)), 3 sheath pump flow rates(0.3, 0.6 and 0.9 mL/min) and 3 backpressure values (120, 150 and180 bar) were tested. The trap employed for flow rate measurementconsisted of a 50 mL Falcon tube containing 20 mL of PEG (Supple-mentary material Fig. 1). Two holes were made in the Falcon tubecap allowing both the admission of the chromatographic mobilephase and the discharge of decompressed CO2. PEEK tubing thatwas 100 mm long and had a 0.250 mm I.D. was directly connectedto the splitter PEEK-sil transfer line and used as a dip tube for mobilephase admission in the Falcon tube. The mobile phase was bubbledinto the PEG, trapping the MeOH, while the exhaust decompressedCO2 came out of the Falcon tube through a stainless steel needle.Every single measurement was performed during 3 min. The wholedevice was weighed before and after each measurement to deter-mine the exact amount of trapped MeOH. This amount was thenconverted into volume and then into flow rate using MeOH densityat the controlled laboratory temperature of 21 ◦C, data are shownin Supplementary material Table 1.

2.3.3. Maximizing MS detection sensitivityDoE experiments were carried out using UHPSFC–ESI-MS/MS

(gradient mode 5–20% of MeOH + 20 mM NH4OH in CO2 (v/v) in

A. Grand-Guillaume Perrenoud et al. / J. Ch

Fig. 2. TIC chromatograms for the separation of 6 drugs obtained with the twoUHPSFC–ESI-MS/MS interfaces. (A) Pre-UV and BPR splitter without sheath pumpinterface (green trace) and (B) Pre-BPR splitter with sheath pump interface (bluet(t

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race). Compounds: (1) Prazepam, (2) theophylline, (3) noscapine, (4) clonazepam,5) papaverine, and (6) indapamide. (For interpretation of the references to color inext, the reader is referred to the web version of this article.)

min at 2 mL/min) in SRM detection mode. For the sake of com-arison, the gradient steepness (product of gradient slope andolumn dead time) was identical for the two other investigatedow rates, i.e., 1.0 and 3.0 mL/min. A mixture of 6 drugs dis-laying different retention times was monitored using the most

ntense SRM transitions, e.g., hydroxyzine (375 → 201), alprazo-am (309 → 281), noscapine (414 → 220), triazolam (343 → 308),apaverine (340 → 202) and methadone (310 → 265). The mixtureas dissolved (100 ppb each) in a ternary injection solvent system

onsisting of EtOH:IPA:heptane, 1:2:7, v:v:v.DoE was also performed using UHPLC–ESI-MS/MS on the same

ixture of 6 compounds dissolved at the same concentrations inure water. The mobile phase consisted of ACN/H2O both contain-

ng 0.1% formic acid (FA), and a gradient run from 15% to 55% ACN in2O (v/v) in 3 min at 0.6 mL/min was employed. The gradient steep-ess was identical for the two other investigated flow rates, i.e., 0.3nd 0.9 mL/min. For both techniques, the same ESI-MS instrumentas used.

. Results and discussion

.1. Characterization of UHPSFC–MS interfaces

.1.1. Evaluation of efficiency, sensitivity and flexibilityBecause of the specific nature of the supercritical mobile phase

nd its high compressibility, well-designed SFC–MS interfaces areequired to avoid loss in chromatographic performance due toobile phase decompression. In the first part of the study, 2 differ-

nt interface configurations were compared based on the followingeatures: preservation of chromatographic integrity and perfor-

ance, detection sensitivity and user-friendliness. To facilitate theisualization, the total ion current (TIC) chromatograms are shownn Fig. 2. The chromatographic parameters and signal intensities ofhe 6 compounds were measured using each individual extractedon current (XIC) and were summarized in Table 1. The first rout-ng interfaces, namely “pre-UV-BPR-split interface” was considered

ainly because its configuration limits as much as possible thextra-column band broadening since the splitter, consisting of aero-dead-volume T-union, is placed prior to the UV detector flow-ell. In addition, the splitter is placed under the direct control

f the active BPR and should provide a relatively good flexibilitys demonstrated in the past in several study [34,35]. The chro-atogram obtained for the separation of the tested drug mixtureith this first interface is reported in Fig. 2A. The efficiency was

romatogr. A 1339 (2014) 174–184 177

measured for each individual compound, and the average N valuewas 8700 plates. A similar separation was obtained using the sec-ond interface, namely “pre-BPR-split + make-up pump interface”(Fig. 2B). Make-up addition of 0.6 mL/min MeOH was not foundto be deleterious to the chromatographic integrity because thebackpressure was still actively controlled by the BPR and sinceno perturbation related mixing effect was observed. In addition,no significant variations in system total pressure (less than 1 bar),retention time (less than 1%) or retention factor (less than 0.5%)were observed between the two configurations, meaning that addi-tional tubing required for the BPR-split + make-up pump interfacehad a negligible effect on separation selectivity. Surprisingly, aslightly improved average N value of 9800 plates was obtained withthis configuration. This result was counterintuitive because, in thissecond configuration, the extra-column volume related to the UV-cell (8 �L) and to the connecting tubes should broaden the peaksdetected in MS since these elements are placed in the same lineas the MS detector whereas they are separated in the first inter-face. This increase in the total system dispersion should logicallyincrease the band spreading and decrease the overall chromato-graphic efficiency of the second configuration compared to the firstone. In the present case, the observed N improvement phenomenonsecond routing interfaces could be explained by the make-up fluidadjunction. Indeed, in both interfaces, the CO2–MeOH mobile phasedecompresses along the MS transfer line (the same dimensions forthe two interfaces). Within this capillary that is no longer underthe direct influence of the BPR, the fluid overall density and inher-ent eluting strength drops, leading to a decrease in the analyte’sspeed. Thus, the analyte detection duration and its inherent peakwidth increase. Nevertheless, using the second interface, the MeOHcomposition of the mobile phase that enters the MS transfer lineis higher due to the presence of additional make-up fluid. Thishigher proportion of MeOH tends to limit the overall density andeluting strength drops of the mobile phase along the transfer capil-lary. In this more “incompressible” mobile phase, the analyte speedwill decrease to a lesser extent than in absence of make-up fluid,leading to shorter detection duration and a thinner peak width. Inthe present case, the efficiency of individual compounds is influ-enced by both higher system dispersion that tends to decreaseN and lower density and eluting strength drops that significantlyincreases N. A significantly better chromatographic efficiency wasobserved using the pre-BPR-split + make-up pump interface forthe 5 more retained compounds (k values between 2.1 and 9.1)which were positively impacted by the lower density and elu-ting strength drops phenomenon and showed an improvement ofefficiency between 8% and 16%. Only the less-retained compound(Prazepam, k = 1.2), which should be the most affected by additionalband broadening, displayed a 5% broader peak (0.020 vs. 0.019 min)with the pre-BPR-split + make-up pump interface compared to thepre-UV-BPR-split configuration. In contrast, the five remainingand more retained drugs. Detection intensity of each individualcompound acquired in SRM mode, the differences between bothsplitting interfaces were noticeable. All 6 compounds displayedhigher peak intensities in the presence of additional make-up fluid.The detection sensitivity was improved by a factor of 5–30 depend-ing on the compounds using the pre-BPR-split + make-up pumpinterface compared to the pre-UV-BPR-split configuration, whilethe background noise remained equivalent. This increase in sensi-tivity was related to the additional MeOH coming from the sheathpump which acted as a supplementary proton source that enhancedthe ionization process.

3.1.2. Chromatographic performance: UHPSFC–UV vs.UHPSFC–MS

The pre-BPR-split + make-up pump configuration was found tobe the best compromise in terms of chromatographic performance,

178 A. Grand-Guillaume Perrenoud et al. / J. Chromatogr. A 1339 (2014) 174–184

Table 1Performance achieved in MS detection using 2 different UHPSFC–MS routing interfaces.

Compound Pre-UV-BPR-split (A) Pre-BPR-split + make-up pump (B)

ka W50%a Na Intensitya ka W50%

a Na Intensitya

Prazepam 1.20 0.019 4240 4450 1.18 0.020 4160 71,600Theophylline 2.16 0.024 6230 249 2.15 0.023 6790 8150Noscapine 2.46 0.024 7200 10300 2.43 0.023 7910 124,000Clonazepam 3.51 0.027 9390 1260 3.47 0.024 12,200 12,300Papaverine 4.43 0.027 13,810 9450

Indapamide 9.62 0.059 11,100 1300

a Values measured based on the MS/MS extracted ion current (XIC).

Fig. 3. Comparison of peak broadening between the UHPSFC–UV configuration (A.,purple trace) and the UHPSFC–ESI-MS/MS configuration using the pre-BPR splitterwith sheath pump interface and on-line UV detector (B., TIC, deep blue trace) andwithout on-line UV detector (C., TIC, light blue trace). Compounds: (1) prazepam,((v

flnadataTccWw

TL

2) theophylline, (3) noscapine, (4) clonazepam, (5) papaverine, and (6) indapamide.For interpretation of the references to color in text, the reader is referred to the webersion of this article.)

exibility and above all detection sensitivity. However, since sig-ificant volumes are added to the system using this configurationnd because of the mobile phase density and eluting strengthrops phenomenon, its influence on chromatographic behaviornd performance needed be further evaluated. For this purpose,he splitter was first completely removed from the instrument,nd the UV detector outlet was connected directly to the BPR.he chromatogram obtained for the mixture of 6 pharmaceutical

ompounds using only UV detection is shown in Fig. 3A and thehromatographic performance values are summarized in Table 2.

ithin this configuration, an average N value of 13,200 platesas obtained. The same separation was performed with MS

able 2oss in chromatographic performance between UV and MS detections.

Compound UV (A) Pre-BPR-split

k W50% N ka

Prazepam 1.14 0.015 6930 1.18

Theophylline 2.09 0.016 13,090 2.15

Noscapine 2.37 0.019 11,000 2.43

Clonazepam 3.41 0.022 14,450 3.47

Papaverine 4.29 0.024 16,700 4.36

Indapamide 9.35 0.047 16,800 9.42

a Values measured based on the MS/MS extracted ion current (XIC).

4.36 0.026 14,970 54,8009.42 0.054 12,850 12,600

detection including the pre-BPR-splitter + make-up pump. Thesplitter inlet was directly connected to the outlet port of the UVdetector, increasing the total extra-column volume by 18 �L (upto 77 �L). Separation selectivity was maintained between the UVtrace (Fig. 3A) and MS trace (Fig. 3B). However, the MS peaksdisplayed in average a 25% lower N value (9800 plates) than thepeaks detected by UV. The first peak (prazepam) was logically themost affected one, but the compounds with longer retention timesstill displayed significant broadening.

The 8 �L UV cell and 10 �L UV detector-BPR connecting tubewere bypassed, and the splitter was directly connected to thecolumn outlet. This third configuration displayed an equivalentextra-column volume than the original UV set-up in Fig. 3A (59 �L).The peak width was expected to be significantly improved usingthis configuration, but surprisingly, an average N value of 10,100plates was measured (Fig. 3C). Only the first peak (prazepam) dis-played a significant plate-count improvement in the absence of theUV flow-cell and connecting tubes. The overall plate count achievedfor this third separation was comparable to that observes in pres-ence of the UV cell and connecting tubes and presented a 23% lowerN value than for the original UV separation. These results corrobo-rate the observations made in Section 3.1.1, which noted the mobilephase decompression phenomenon as the major contributor forband spreading in the MS configuration.

3.2. Modeling of splitter interface behavior

3.2.1. Influence of chromatographic parametersDue to its design, the operation of the pre-BPR-split + make-up

pump configuration is directly under the influence of chromato-graphic parameters. The simplest way to understand the principleof operation of this interface was to measure the amount of mobilephase per time unit that is actually directed toward the ESI probe.Because the CO2 is passing from a homogenous condensed phaseto a gaseous state while running across the transfer capillary, theonly measurable parameter is the remaining MeOH content thatcan be collected at the capillary outlet. A homemade MeOH trap

preventing the potential evaporation of liquid MeOH was designedand directly connected to the transfer line outlet in place of theESI probe. Eighty-one different chromatographic conditions wereused to assess the influence of backpressure, flow rates (SFC and

+ make-up pump with UV (B) Pre-BPR-split + make-up pump without UV (C)

W50%a Na ka W50%

a Na

0.020 4160 1.18 0.017 55700.023 6790 2.15 0.023 67400.023 7910 2.45 0.022 82200.024 12,200 3.50 0.024 12,5900.026 14,970 4.40 0.026 14,9800.054 12,850 9.42 0.054 12,940

A. Grand-Guillaume Perrenoud et al. / J. Chromatogr. A 1339 (2014) 174–184 179

F r witha eOH)

d 0 bar

moohtmItspccotflhtta

FU

ig. 4. Model of total MeOH amount entering the ESI probe using the pre-BPR splittend sheath pump flow rate (y-axis) for two different UHPSFC mobile phase (CO2/Mifferent fixed backpressure values, 120 bar (A1 and B1), 150 bar (A2 and B2) and 18

ake-up pumps) and SFC mobile phase composition variationsn the amount of MeOH collected per time unit. First, preliminarybservations showed that an increase in the backpressure lead to aigher amount of collected MeOH. This effect is predictable becausehe higher resistance generated by the BPR obviously redirects a

ore important part of the mobile phase toward the transfer line.n the same way, an increase in the make-up pump flow rate or inhe SFC mobile phase MeOH content evidently extended the mea-ured amount of MeOH in the trap. In contrast, faster SFC mobilehase flow rates with constant MeOH composition led to a signifi-ant reduction in MeOH directed toward the transfer capillary. Thisounterintuitive effect is related to the fact that the active BPR legf the splitter compensates pressure generated by the mobile phaseo maintain a constant outlet backpressure. In the event of higherows, the BPR is more open and offers a path of least resistance and

ence the MS capillary receives less flow. Now, in contrast, whenhe mobile phase flow rate is low, the BPR must close in ordero maintain system pressure, hence generating greater resistancelong that leg and send more flow through the MS capillary.

ig. 5. Model of the split ratio (A) and UHPSFC mobile phase dilution factor caused by theHPSFC mobile phase flow rate (x-axis) and sheath pump flow rate (y-axis) for a fixed UHPS

sheath pump interface as a function of the UHPSFC mobile phase flow rate (x-axis)compositions, 95/5 (v/v) for (A) surfaces and 80/20 (v/v) for (B) surfaces, and three

(A3 and B3).

Based on these preliminary observations and considering thetubing dimensions and Eq. (1), the MeOH flow rate entering theESI probe was modeled over the whole range of chromatographicconditions using Excel. Some examples are shown in Fig. 4, includ-ing different mobile phase compositions (CO2/MeOH: 95/5 (v/v) for(A) surfaces and 80/20 (v/v) for (B) surfaces) and various backpres-sures (120, 150 and 180 bar for surfaces 1, 2 and 3, respectively).As shown, the shape of these surface responses confirms the pre-vious experimental observations. The validity of the model wasthen experimentally verified with 12 randomly selected sets of flowrates, backpressures and an extended set of mobile phase compo-sitions (between 2% and 40% MeOH in CO2 (v/v)). Less than 10%difference was measured between the amount of MeOH physicallycollected in the trap and the value predicted by the computer sim-ulation, which can therefore be considered as valid. In addition, it

is worth mentioning that thanks to the dynamic adaptation of thesplit ratio between transfer capillary and the BPR depending on thechromatographic conditions, the total flow rate of MeOH enteringthe ESI probe tends to be leveled. The MeOH flow rate is always

sheath flow (B) for the pre-BPR splitter with sheath pump interface as a function ofFC mobile phase (CO2/MeOH) composition of 90/10 (v/v) and 150 bar backpressure.

180 A. Grand-Guillaume Perrenoud et al. / J. Chromatogr. A 1339 (2014) 174–184

Table 3Investigated levels of the variables involved in Design of Experiment (DoE) methodology.

Level UHPSFC ESI

SFCF (mL/min) BPR (bar) SheaF (mL/min) CapV (kV) DesoT (◦C) DryF (L/h)

babapMtEp

3

cwipa(dfltBhRBsaswstbtbadptthpdaucp

3

sacdcch

not explain ESI-MS sensitivity (confidence interval crosses zero).These parameters do not affect the concentration of the analytesin the mobile phase or the ESI spray stability because our splitter

Fig. 6. Detection sensitivity optimization. Screening the most important operatingparameters in UHPSFC–ESI-MS/MS and their influence on detection sensitivity ofbasic drugs: noscapine (deep blue), papaverine (green), hydroxyzine (gray), alpra-

−1 1.0 120 0.3

0 2.0 150 0.6

+1 3.0 180 0.9

etween 125 and 300 �L/min when typical operating conditionsre applied in both isocratic and gradient modes (e.g., SFC flow rateetween 1.0 and 3.0 mL/min, make-up pump flow rate between 0.3nd 0.6 mL/min, backpressure between 120 and 150 bar and mobilehase compositions between 2% and 40% MeOH in CO2 (v/v)). ThiseOH flow rate range is well suited to preventing analyte precipi-

ation inherent to CO2 decompression and to ensure both optimalSI spray formation and good proton transfer during the ionizationrocess.

.2.2. Split ratio and band dilution factorThe passive changes in the split ratio related to chromatographic

onditions within the pre-BPR-split + make-up pump interfaceere further characterized by our computer simulation. Fig. 5A

llustrates the amplitude of the split ratio for different SFC mobilehases and make-up flow rates at a fixed backpressure of 150 barnd a constant mobile phase composition of CO2/MeOH: 90/10v/v). A split ratio between 2 and 8 between the mobile phaseirected toward the BPR and the MS is expected depending on theow rate settings. Lower backpressure value or higher MeOH con-ent tend to further direct the mobile phase in the direction of thePR (split ratio up to 12) due to lower resistance from the BPR origher generated pressure within the transfer line, respectively.egarding MS detection sensitivity, a high split ratio toward thePR could constitute a significant concern in the case of a mass-flowensitive ionization technique such as APCI. Because only a limitedmount of analyte would be directed to the MS probe, the loss ofensitivity expected would be proportional to the split ratio butould be less than 1 order of magnitude. In contrast, the detection

ensitivity achievable with a concentration-dependent ionizationechnique such as ESI would not be affected by the split ratioecause flow division has no effect on the analyte concentration inhe mobile phase. Nevertheless, detection sensitivity in ESI coulde affected by make-up fluid adjunction into the mobile phase. Thedditional amount of MeOH from the sheath pump introduces ailution factor that reduces the analyte concentration in the mobilehase. This dilution factor was plotted for different flow rate condi-ions in Fig. 5B and was found to be between 1.1 and 3.5. However,he latter high value is only observed in “exotic” conditions where aigh sheath pump flow rate (1.0 mL/min) is added to a low mobilehase flow rate (0.4 mL/min). Under more typical conditions, theilution factor was much more reasonable and varied between 1.1nd 1.5. Finally, it is worth mentioning that using a constant make-p flow rate when performing a separation in gradient mode willause the later eluting peaks to be more diluted than less retainedeaks.

.3. Maximizing MS detection sensitivity

This section focuses on the influence of various SFC and ESIource parameters on MS detection sensitivity. To be routinelypplicable, these parameters and their ranges of investigation werehosen taking into account additional constraints related to con-

itions of UHPSFC–ESI-MS/MS for the analysis of pharmaceuticalompounds. Thus, the nature of the mobile phase (MeOH) andolumn dimensions (3.0 mm × 100 mm, 1.7 �m) were fixed before-and in order to be as representative as possible of UHPSFC–MS

+1.0 200 300+2.5 325 650+4.0 450 1000

daily use. The impact of chromatographic and ionization parame-ters on sensitivity was assessed in UHPSFC–ESI-MS/MS, using thepre-BPR-split + make-up pump interface following a 2-step design-of-experiment (DoE) methodology. In parallel, the optimization ofanalytical parameters was also performed on the same ESI-MS/MSplatform hyphenated to the UHPLC system. The constraints relatedto latter instrument have been taken into account in selectingthe dimensions of the chromatographic column (2.1 mm × 50 mm,1.7 �m) and representative mobile phase conditions for RPLC anal-ysis for pharmaceutical molecules were preferred. Finally, theabsolute detection sensitivity performance obtained on the twoindividually optimized analytical platforms were compared.

3.3.1. Screening for the most important operating factors inUHPSFC–ESI-MS/MS

First, the critical experimental variables were selected for the ESIionization source (i.e., capillary voltage (CapV), desolvation tem-perature (DesoT), drying gas flow rate (DryF)) and UHPSFC (i.e.,mobile phase flow rate (SFCF), backpressure value (BPR) and sheathpump flow rate (SheaF)). The investigated range for each variableis summarized in Table 3, and lower/higher levels were chosen tocover a sufficiently wide but rational range of operating conditions.The peak intensities of the 6 basic model compounds were selectedas the analytical response. A half-fractional factorial design (HFFD)was selected, and the central conditions were repeated 6 times toestimate the experimental error. A total of 22 runs (16 for HFFD and6 central points) were randomly carried out.

The model coefficients for the 6 parameters, including 95% con-fidence intervals, are presented in Fig. 6. The coefficient size anddirection (toward a positive or negative value) represent the effecton detection sensitivity when a single operation parameter variesfrom 0 to 1, while the 5 remaining factors are maintained at theiraverage values. As expected, the SFCF and BPR parameters did

zolam (purple), triazolam (light blue) and methadone (red). Parameters: capillaryvoltage (CapV), desolvation temperature (DesoT), drying gas flow rate (DryF), sheathpump flow rate (SheaF), UHPSFC mobile phase flow rate (SFCF), backpressure (BPR)and UHPLC mobile phase flow rate (LCF). (For interpretation of the references tocolor in text, the reader is referred to the web version of this article.)

A. Grand-Guillaume Perrenoud et al. / J. Chromatogr. A 1339 (2014) 174–184 181

F AlpraD

tEiacpssot

3

dcfipaawaamcadpofveit4t(i

csdrt(

ig. 7. Ionization source conditions optimization. Detection sensitivity modeling forrying gas flow rate was fixed at 1000 L/h in these representations.

ends to ensure regular and sufficient mobile phase flow toward theSI source. Regarding the SheaF, a small but significant decreasedntensity effect was highlighted when the make-up pump delivered

higher flow rate. This was not surprising because higher MeOHontent tends to dilute the analyte concentration in the mobilehase. Thus, a low SheaF should be selected to limit the loss ofignal intensity. Finally, ESI variables were shown to play the mostignificant roles for detection sensitivity, with a positive influencef high DesoT and DryF and a negative influence of CapV, and wereherefore selected for further investigations.

.3.2. Sensitivity modeling and optimizationTo optimize the values for the three most influent parameters on

etection sensitivity (i.e., CapV, DesoT and DryF), a face-centeredentral composite design (CCD) was selected. The explored rangesor the three ESI parameters remained identical to those reportedn Table 3, while the chromatographic parameters for the mobilehase flow rate and the BPR were held constant at 2.0 mL/minnd 120 bar, respectively. The make-up pump flow rate, whichlso affects the response, was set at 0.3 mL/min to limit solventaste and attain reasonable sensitivity. Experimental error was

gain estimated using 6 additional trials under average conditions,nd a simple statistical study showed acceptable repeatability. Theodel validity was judged sufficient because the determination

oefficients (R2) of the response surface models were between 82%nd 91% after removal of one outlier trial. The models displayedifferent intensity amplitudes depending on the investigated com-ound but exhibited the same response surface shape regardlessf the analyte. As an example, Fig. 7A illustrates the response sur-ace model obtained for alprazolam with DryF fixed at its optimalalue, i.e., 1000 L/h. The curved shape clearly reflects an interactionffect between parameters. For all compounds, the best sensitiv-ty response was achieved at the lowest CapV value of 1.0 kV andhe highest investigated DryF and DesoT values, i.e., 1000 L/h and50 ◦C, respectively. Investigations of more extreme values (towardhe lowest CapV (<1.0 kV) and highest DryF (>1000 L/h) and DesoT>450 ◦C)) could not be tested due to instrumental limitations ornstability.

For a fair comparison of detection sensitivity, the experimentalonditions were also optimized to achieve the highest possible sen-itivity in the UHPLC–ESI-MS/MS configuration using the same MS

etector. Taking into account the UHPLC column dimensions, sepa-ations were performed at 0.6 mL/min to achieve fast analysis closeo the optimal linear velocity. The effect of ionization parametersCapV, DryF and DesoT) on detection sensitivity was determined

zolam using the UHPSFC–ESI-MS/MS (A) and UHPLC–ESI-MS/MS (B) configurations.

using a methodology similar to that employed in UHPSFC–ESI-MS/MS. Fig. 7B shows the response surface model for detectionsensitivity achieved in UHPLC–ESI-MS/MS for alprazolam (DryFfixed at its optimal value, i.e., 1000 L/h). Interestingly, the responsesurfaces obtained in UHPLC–ESI-MS/MS conditions were very closeto those achieved in UHPSFC–ESI-MS/MS, only slight changes in theshape curvatures were observed. Moreover, the optimal conditionsfor maximizing sensitivity were rigorously identical between bothtechniques. Initially, this observation is surprising since the compo-sition of mobile phase is very different between UHPSFC and UHPLC(i.e., CO2/MeOH vs. H2O/ACN, respectively). However, the fact thatCO2 decompresses in the transfer capillary under the UHPSFC con-ditions must be kept in mind. Then, its solvating power is severelydecreased, and analytes are certainly only solubilized in liquidMeOH when they reach the ESI source before being ionized. Theseconditions are close to those encountered in liquid chromatogra-phy conditions, when molecules are dissolved in a hydro-organicliquid mixture. The requirements for nebulization processes (dry-ing gas and desolvation temperature) must therefore be similar. Forthe latter parameter, the need for a high temperature to evaporatethe aqueous part of the hydro-organic mobile phase in UHPLC isobvious, but it might be more surprising for the UHPSFC mobilephase. Most likely, a high DesoT is required to compensate for thelow MeOH temperature inherent to CO2 decompression, which isa strongly endothermic process.

3.3.3. Sensitivity comparisonThe most notable difference between UHPLC and UHPSFC

is obviously the solvent composition of the mobile phases(MeOH/CO2 vs. hydro-organic mixture). Although this compositionhas no influence on the optimal ESI settings, as shown previously,it is highly probable that it impacts the efficiency of ionization andespecially the nebulization processes that further govern detectionsensitivity. This phenomenon can be illustrated within the samechromatographic technique when comparing the peak intensityachieved with the best ESI conditions to that obtained when theworst ionization settings determined with our model are applied.Fig. 8 shows the chromatograms obtained with the mixture of6 basic drugs achieved by UHPSFC (traces A) and UHPLC (tracesB). The top traces (red; A1 and B1) were obtained using the bestESI conditions, i.e., 1.0 kV, 1000 L/h and 450 ◦C for CapV, DryF

and DesoT, respectively; middle traces (green; A2 and B2) wereobserved using average source conditions, i.e., 2.5 kV, 650 L/h and325 ◦C for CapV, DryF and DesoT, respectively; and bottom traces(blue; A3 and B3) were achieved using the worst ESI settings i.e.,

182 A. Grand-Guillaume Perrenoud et al. / J. Chromatogr. A 1339 (2014) 174–184

Fig. 8. Detection sensitivity (TIC) changes as a function of ionization parameter variations for UHPSFC–ESI-MS/MS (A: traces) and UHPLC–ESI-MS/MS (B: traces) configurationsusing a mixture of basic drugs ionized at optimal source conditions (A1 and B1 (red traces)), at average source conditions (A2 and B2 (green traces)) and at the worst sourcec hydror

4iltUspTfmtRwwSp4ls5tTmimwthwo

mdettl(s

onditions (A3 and B3 (blue traces)). Compounds: (1) noscapine, (2) papaverine, (3)eferences to color in text, the reader is referred to the web version of this article.)

.0 kV, 300 L/h and 250 ◦C for CapV, DryF and DesoT, respectively. Its worth mentioning that the SRM baseline noise remained veryow (<100 counts) within the different ESI settings of the sameechnique and between both chromatographic techniques. ForHPSFC–ESI-MS/MS (traces A), a decrease in the overall detection

ensitivity between a factor of 8 and 14, depending on the com-ound, was observed between the best and worst ESI conditions.his sensitivity decrease was significant but remained limited. Aactor of 1.5–2 was attributed to the impact of CapV, whereas the

ain sensitivity decrease was due to the nebulization process andhe DryF and DesoT settings (factor comprised between 7 and 12).egarding UHPLC–ESI-MS/MS (traces B), a severe loss of sensitivityas observed when the worst conditions were applied. Papaverineas not detected, and the 5 remaining compounds displayed a poor

/N ratio (<10). When optimal and worst ESI settings were com-ared, the peak intensity deterioration was found to be between5 and 215 times in UHPLC, depending on the compound. Simi-

arly to UHPSFC–ESI-MS/MS, the CapV value variations were onlylightly responsible for the sensitivity loss (factor between 2 and). The nebulization process was identified as the major contribu-or to signal loss (decrease factor comprised between 10 and 35).his significant difference in the nebulization process efficiencyay be because decompressed CO2 gas acts as an additional dry-

ng gas at the ESI tip with the UHPSFC mobile phase. However, itay most likely be due to the presence of a large proportion ofater in the UHPLC mobile phase, which requires a high tempera-

ure and gas flow to be properly evaporated. These results clearlyighlight the robustness of the UHPSFC–ESI-MS/MS configuration,hich maintains acceptable detection sensitivity over a wide range

f ESI conditions.The absolute detection performance obtained using each chro-

atographic approach under optimized conditions were thenirectly compared. Because the injected concentrations and thexperimental SRM background noise were equivalent for bothechniques, the comparison was performed on the intensity of

he extracted peaks. Fig. 9 shows the XIC of the 6 basic ana-ytes obtained using the optimal conditions in UHPSFC–ESI-MS/MStraces A) and UHPLC–ESI-MS/MS (traces B). Peak intensity variedignificantly depending on the compound and on the analytical

xyzine, (4) alprazolam, (5) triazolam, and (6) methadone. (For interpretation of the

approach. However, the overall quantitation limit was always inthe range of 0.1–20 ng/mL. For 5 compounds, significantly bet-ter detection sensitivity (enhancement factor between 4 and 10)was observed by UHPSFC–ESI-MS/MS vs. UHPLC–ESI-MS/MS. Thehigher efficiency of the desolvation process for compounds presentin the methanolic effluent (UHPSFC) compared to those solubilizedin a hydro-organic mobile phase (UHPLC) is certainly responsiblefor this finding. Methadone was the only molecule that displayeda stronger response in UHPLC–ESI-MS/MS (10 times more sensi-tive than in UHPSFC–ESI-MS/MS). Chromatographic issues couldpartially explain this observation. Suitable peak shape may be dif-ficult to achieve under supercritical conditions for relatively strongbasic compounds (pKa > 8.0). NH4OH adjunction within the mobilephase was sufficient to limit peak shape degradation such as tailingor fronting but could be inefficient to avoid peak broadening relatedto secondary interactions with the polar chromatographic support[32]. Methadone possesses this critical characteristic of a high rangebasic pKa value (≈9.1) and furthermore displays high retention inUHPSFC. Its W50% achieved in gradient mode under supercriticalconditions was measured to be 25% broader than the 5 other peaks,whereas it remained equivalent to the others in liquid conditions.Peak broadening reduces methadone peak height and detectionsensitivity. Another probable explanation is due to the fact thatmethadone exhibits higher retention in liquid conditions and thusrequires higher ACN proportions (approximately 50%) to be elutedfrom the UHPLC support. Higher ACN content has been described tohave a positive effect during the nebulization step, leading to betteranalyte desolvation, which could further enhance detection sensi-tivity [36]. Finally, it should be noted that the injected volumes setfor UHPLC and UHPSFC were not scaled in proportion to the columnvolumes in both techniques. Indeed, the chromatographic sup-port used in supercritical conditions (100 mm × 3.0 mm, 1.7 �m)possesses a 4-times larger volume than the narrow-bore column(50 mm × 2.1 mm, 1.7 �m) mounted in the UHPLC setup. In thepresent case, the injected volume was held constant at 1 �L to

limit peak shape issues caused by a large injected volume in SFC[37] and to avoid repeatability problems that could occur when theinjected volume is strongly reduced (<1 �L) in LC. A further increasein detection sensitivity in UHPSFC–ESI-MS/MS by a factor of 4 might

A. Grand-Guillaume Perrenoud et al. / J. Chromatogr. A 1339 (2014) 174–184 183

Fig. 9. Comparison of the relative detection sensitivity (XIC) of basic drugs between UHPSFC–ESI-MS/MS (A traces) and UHPLC–ESI-MS/MS (B traces) configurations at optimalc yzine( the we

ta

4

IptoaiugecflmmaiptmtvCc

tmtflvMtsmc

onditions. Compounds: noscapine (deep blue), papaverine (B green trace), hydroxred). (For interpretation of the references to color in text, the reader is referred to

hus be expected if the injection volume could be proportionallydjusted.

. Conclusion

The hyphenation of SFC with MS is not obvious at first glance.ndeed, the unique nature of the supercritical mobile phase andressure constraints required to maintain its physical proper-ies may appear to be major limitations to the implementationf such a platform. However, the use of a suitable hyphen-tion interface makes SFC–MS straightforward. Among the variousnterfaces tested in the present study, the pre-BPR-split + make-p pump interface clearly outperforms the others by offeringood flexibility in terms of applicable chromatographic conditions,xtended robustness and ease of use. Its design is well suited for aoncentration-sensitive ionization technique such as ESI becauseow splitting does not affect analyte concentration within theobile phase. In addition, this configuration allows the control ofobile phase properties due to the downstream BPR, while the

ddition of a make-up solvent helps avoid analyte precipitationnherent to CO2 decompression and enhance the ESI ionizationrocess efficiency. Compared to the UV configuration, the split-ing interface does not disrupt the chromatographic integrity and

aintains separation selectivity. Nevertheless, an average reduc-ion of 25% in chromatographic efficiency was observed in MSs. UV detection. This loss was attributed to the post-splitterO2 decompression phenomenon rather than to additional extra-olumn volume.

A computer simulation program was built to better understandhe operation mechanism of the particular splitting interface. This

odel allows the accurate simulation over a large set of condi-ions of the influence of different chromatographic parameters (i.e.,ow rate and mobile phase composition, flow rate of make-up sol-ent or backpressure) on the amount of MeOH directed toward theS detector. The model clearly shows that the flow sent toward

he MS source remains in the ideal range for the formation andtability of the ESI spray (125–300 �L/min), regardless of the chro-atographic conditions. Moreover, even if the latter drastically or

ontinuously varies, e.g., gradient mode separation, the interface is

(gray), alprazolam (B purple trace), triazolam (B light blue trace) and methadoneb version of this article.)

able to dynamically adapt its split ratio to minimize the impact onthe final flow directed toward the ESI source. In addition to the splitratio, the simulation also provides a clear idea of the dilution factorinduced by make-up solvent adjunction and shows that it has a rea-sonable impact (less than 1.5 times dilution) when reliable UHPSFCconditions are applied.

The sensitivity of MS detection was maximized for UHPSFC–ESI-MS/MS using a chemometric approach. The effects and significanceof 6 operating factors related to chromatographic conditions andthe evaluation of the ESI settings were determined to achieve ahigh detection sensitivity of a mixture of basic drugs. The resultsshowed that the ESI-related parameters, namely CapV, DryF andDesoT, were the most significant variables. It appears that a lowCapV should be associated with high DesoT and elevated DryF toachieve the highest sensitivity. The same conclusions were alsodrawn for the UHPLC–ESI-MS/MS conditions.

Finally, better sensitivity (factor 4–10) was obtained forUHPSFC–MS vs. UHPLC–MS in most cases. This improved sensitiv-ity was attributed to better efficiency of the analyte desolvationprocess with SFC effluent consisting of liquid MeOH surrounded bydecompressed CO2.

Acknowledgments

The authors wish to thank Hélène Boiteux and Dr. IsabelleFranc ois from Waters for stimulating discussions and valuablecomments and advice. Many thanks also to Joël Fricker from Watersfor the loan of the make-up pump. Cecilia Romano from the Uni-versity of Torino is acknowledged for performing the experimentspresented in Figs. 8 and 9. Many thanks also to Bertrand Duléry andJoëlle Verne from Sanofi-Aventis for contributing to the develop-ment of the idea and financial support.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.chroma.2014.03.006.

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