RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 8,333-338 (1994)

Analysis of Neuropeptides by Perfusion Liquid ChromatographylElectrospray Ion-trap Mass Spectrometry Hung-Yu Lint and Robert D. Voyksner* Analytical and Chemical Sciences, Research Triangle Institute, PO Box 12194, Research Triangle Park, North Carolina 27709, USA

Perfusion high-performance liquid chromatography (HPLC) combined with electrospray ion trap mass spec- trometry (ITMS) was evaluated for the determination of neuropepetides in plasma. Perfusion HPLC offers the capability of resolving neuropeptides spiked into plasma in 5 min compared to the 30-60 min separations performed on packed capillary CIS columns. Electrospray combined with the ITMS provides the ability to ionize these neuropeptides and mass analyze them with high sensitivity and specificity. Sub-picomole quantities of neuropeptides injected on-column could be specifically detected in a plasma matrix. The electrospray-ITMS mass spectrum of each neuropeptide showed multiply charged ions which could be used to determine or confirm their molecular weights.

Neuropeptides are involved in intercellular communi- cation in a variety of biologically important functions such as muscular control, metabolism, reproduction, and other activities.',' It is hence important to identify these compounds and their metabolites both qualitati- vely and quantitatively. Analysis of neuropeptides has been reported by off-line HPLC fast-atom bombard- ment mass spe~t rometry ,~-~ and by capillary electrophoresis/electrospray mass spectrometry.6 However, the low picomole to femtornole levels of neuropeptides in biological fluids as well as the com- plexity of the matrices make it very difficult and time- consuming to purify these compounds sufficiently for their analysis. The recent development of electrospray coupled to mass spectrometers allows for the produc- tion and determination of intact ions from these non- volatile, thermally labile peptides, whose m/z values usually fall within the range of most mass anlayzers.

Electrospray is an ionization method which can effi- ciently desorb ions formed in solution into the gas phase for mass a n l a y ~ i s . ~ - ~ A high electric field applied to the atmospheric-pressure spray chamber results in the initial nebulization and charging of the LC effluent from the spraying needle. The droplets are continu- ously desolvated by a heated counterflow of nitrogen bath gas. When the droplet is desolvated, the radius of the charged droplets decreases and the field density increases, resulting in the analyte desorbing from solu- tion into the gas phase as an intact ion (ion evaporation) .lo Depending on the solute species, its molecular weight and its ability to acquire charge, the ion evaporation process can generate multiply charged gas-phase ions, which effectively extends the mass range of mass spectrometers, enabling the analysis of neuropeptides by mass spectrometry.

ITMS has been shown to provide superior sensitivity over quadrupole instruments due to the ability to accumulate ions in the trap and to sample ion currents for much longer than in a scanning quadrupole mass

'Present address: Sterling Winthrop Inc., 9 Great Valley Parkway, Malvern, PA 19355, USA.

Author for correspondence.

analyzer (high duty cycle). Also there are minimal losses of ions in the trap during accumulation and mass analysis compared to a quadrupole. The ITMS can also be operated in the tandem (MS") mode to generate additional structural inf~rmation."-'~ Initial investi- gations using an electrospray interface combined with an ion trap have shown its utility with a variety of samples. l4-I8 These advantages of electrospray and ITMS can be used to achieve sensitivities greater than those obtained by other LC/MS technique^.'^

The advantages of on-line HPLC/MS and the high sensitivity of electrospray-ITMS have prompted the use of on-line capillary HPLC/electrospray-ITMS for the analysis of neuropeptides. Capillary HPLC techniques have advantages with regard to chromatographic reso- lution and sensitivity when working with limited quanti- ties of samples.2".21 Sharp chromatographic peaks, together with minimal column adsorption, offer the potential to detect trace levels of neuropeptides in complex matrices. Furthermore, flow rates typically used for capillary separation (1-10 pL/min) are com- patible for direct coupling with electrospray mass spec- trometry. Recently, separation times have been greatly reduced through the use of perfusion HPLC.22-26 Perfusion columns are capillary columns packed with derivatized polystyrene divinylbenzene which has particle-through pores of 6000-8000 A diameter inter- connected by 300-1500 A diffuse pores.23 Perfusion columns can be operated at 10-20 times higher flow rates than conventional HPLC columns, reducing sep- aration time accordingly.

The coupling of perfusion chromatography with elec- trospray mass spectrometery has been d e m o n ~ t r a t e d . ~ ~ . ~ ~ The 40-80 pL/min flow rate from a capillary perfusion column could be accommodated using an ion spray (pneumatically assisted electrospray) i n t e r f a ~ e ~ - ~ ' or using an ultraspray (ultrasonic nebuliz- ation) i n t e r f a ~ e . ~ ' - ~ ~

This paper details the use of perfusion capillary HPLC with electrospray ITMS for the determination of neuropeptides in plasma. The capabilities of capillary perfusion HPLC with electrospray ITMS will be high- lighted.

Received 4 November 1993 Accepted (revised) 12 February 1994

CCC 0951-4198/94/040333-06 0 1994 by John Wiley & Sons, Ltd

334 PERFUSION HPLC/ELECTROSPRAY-ITMS OF NEUROPEF'TIDES

EXPERIMENTAL

Standards and samples

Neuropeptides (adrenocorticotropic hormone (ACTH), dynorphin A , dynorphin B, a-endorphin, /?-endorphin, a-melanocyte stimulating hormone (a- MSH)), arginine, and gramicidin S were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The neuropeptides were dissolved in 20% acetonitrile (ACN) (Baxter, Muskegon, MI, USA) in water with 0.1% trifluoroacetic acid (TFA) or 0.1% formic acid. Canine blood (8-9 mL) samples were centrifuged at 10 000 rpm for 10 min and 250 pL aliquots prepared by solid-phase (Cis) extraction. The spiked plasma sam- ples were prepared by mixing the standard neuropep- tide solutions to the solid-phase extract to achieve the desired concentration (less than 10 pL of standard solu- tion containing the neuropeptides was spiked into the 250 pL plasma extract).

Electrospray-ITMS hardware and optimization

An ITMS (Finnigan MAT, San Jose, CA, USA) was coupled to an electrospray interface (Analytica of Branford, Branford, CT, USA) in a manner previously described.33 Specifically the tubular exit lens of the electrospray was designed so that the 0.d. of the lens could snugly fit the i.d. of the end-cap cavity of the filament assembly to ensure alignment of the electro- spray with the ion entrance hole in the end cap. The tubular lens was electrically isolated from the end cap, using Teflon tape, to provide the ability to gate ions into the trap. An adjustable gating circuit was built to vary the gating voltages. A gating voltage of -45 V was used to introduce ions into the ion trap while +20V was used to prevent ions from entering the ion trap. These voltages were toggled using the output of the ITMS gate control for electron ionization (EI) oper- ation.

The Analytica electrospray interface employed a counterflow of nitrogen gas heated to 200 "C arid intro- duced at a flow rate of about 300 mLlmin to desolvate ions formed at atmospheric pressure. This heated coun- terflow gas was essential to obtain a stable ion current. The electrospray atmospheric chamber voltages and needle position, electrospray transport skimmer and lens voltages, and the ITMS parameters were opti- mized by monitoring the [M+H]+ ion of arginine (mlz 175) and the [M+2Hl2+ ions of gramicidin S (mlz 572). This 'tuning' solution containing 200 pglmL of arginine and 100 pg/mL gramicidin S in 50l50 aceto- nitrile (ACN) + H 2 0 with 0.1% TFA was infused into the electrospray system at 2 pLlmin. Fine adjustment of the system was performed at the beginning of the daily operation. The optimized parameters were then used throughout the experiments: Electrospray : Cylindrical electrode V,

End plate electrode V2 Capillary entrance V3 Capillary Exit Skimmer 1 Skimmer 2 Lens 1 Lens 2 Gate (Lens 3)

-2900 V -3200 V - 3900 V

150 V 37.9 v 3.2 V

-73.2 V -98.4 V -45.4 v

ITMS: Ionization time 70 ms at a qL of 0.15 Desolvation time 0 or 100ms Resonance ejection qL 0.23 Resonance ejection voltage 6 V Pressure with helium 4.0 X Torr Background pressure with

electrospra y 1.6 x Torr

Mass analysis on the ITMS The mass selective instability mode34 was used to eject ions from the ion trap to the multiplier for detection. An ion with a specific mlz value can be efficiently trapped as determined by the Mathieu stability diagram.35 When the Mathieu parameter qL of an ion reaches 0.908, it will be ejected. By scanning the amplitude of the RF voltage applied to the ring elec- trode, ions with different mlz values are sequentially ejected and detected.

By varying the scan function parameters in the ITMS, a supplementary R F frequency can be chosen to excite ions and force them to exit the ion trap at much lower values of qz than 0.908. This method (resonance ejection) was used to extend the detectable range of mlz of the ITMS to 2600.36-38 Mass calibration for resonance ejection was conducted by performing a linear regression of the measured mlz values versus the calculated mlz values of arginine, gramicidin S, a- endorphin, or P-endorphin. The plot gave a correlation factor (R) of 0.99 for a 3-point calibration. With this established calibration plot, the calculated molecular weight of a compound was within f 0 . 2 D a of the calculated value. The calibration was checked period- ically with standard solutions. Long-term accuracy of the calibration was within f 0.5 Da when operating at a pressure 4 x Torr and when ioni- zation times were sufficiently short to prevent space charging. The trap was recalibrated if the mass error for the standard was > f 0.5 Da. Recalibration typically occurred when operating under different pressure and ion gating times than the original calibration or when the RF electrode wire was adjusted in length or when the trap was cleaned.

Torr f 0.2 x

HPLClMS Two Waters 6000A HPLC pumps (Waters Inc., Milford, MA, USA) provided a 0.2mLlmin solvent flow to a tee splitter. A 15 cm x 2.1 mm CI8 column was connected to one end of the splitter. The other end of the splitter was attached to a Rheodyne 8125 injector (Rheodyne Inc., Cotati, CA, USA) with 0.5 pL sample loop followed by a 15 cm x 0.32 mm ClS 300 A column (LC Packings Inc., San Francisco, CA, USA) with 5 pm particles. A 50 pm i.d. deactivated fused silica capillary connected the column through an ISCO CV4 variable wavelength absorbance detector (ISCO Inc., Lincoln, Nebraska, USA) equipped with a capillary electrophoresis flow cell to the electrospray needle assembly. This balanced column split allowed a flow rate of about 4 pL/min to the electrospray interface. Separations were performed with a gradient of 5% acetonitrile to 100% acetonitrile in water with 0.05% TFA in 50min with a hold at the final condition for 10 min and an equilibration time of 30 min.

The perfusion chromatography was performed using

PERFUSION HPLC/ELECTROSPRAY-ITMS OF NEUROPEPTIDES 335

loo

.- g a, c - ; 50 ._ c 0 -

the same HPLC equipment with a 'C,-like' 15cmx 0.32 mm 300 8, diffusive porous, 6000-7000 8, through pores POROUS I1 R /H perfusion column (LC Packing) instead of a C18 column. Operating the HPLC pump at 1.5 mL/min with the balanced column split resulted in a flow rate of -35 yL/m in through the perfusion column. The separation of the neuropeptides was performed using a gradient of 5% to 70% acetoni- trile in water with 0.1% formic acid in 4 min and then hold for 2min. The.solvent flow was maintained for 5 min before each analysis to ensure equilibration to initial solvent conditions. The eluate from the perfusion column was introduced into the electrospray interface (equipped with a capillary shield initially described by Whitehouse and co-workers,' to enhance desolvation in the atmospheric-pressure chamber). This capillary shield focuses the counterflow nitrogen gas in front of the capillary entrance to aid in desolvation and limits the sampling of clusters and droplets that can con- tribute to spike noise at flow rates of 30-40 pL/min.

(c) 1734 [M+ZH] *+

[M+3HI3+ 1156

RESULTS AND DISCUSSION Optimization of electrospray-ITMS Optimization of the Analytica electrospray interface with the ITMS involved the evaluation of various ITMS parameters that affect overall sensitivity for gramicidin S and arginine. While these parameters primarily in- volve conditions for ion injection (ionization time, q2, and helium pressure), desolvation and ion transmission into the ITMS were also studied. Initially the diameter of the orifice in the entrance end-cap was varied to maximize ion transmission into the ion trap. Increasing the diameter from 1 mm (standard size) to 3mm resulted in a three times increase in signal. However, increasing the diameter to 4mm or larger resulted in decreased sensitivity and a mass shift possibly due to the space-charge effect and the unsuitable trapping fields inside the ion trap. Ion injection into the trap was optimal at a total pressure of 4X lO-,Torr (1.6 x lo-, Torr nitrogen from the electrospray inter- face with the addition of helium to reach 4 X lo-, Torr pressure uncorrected ion gauge reading) due to colli- sional damping. Lower pressures resulted in a decrease in signal particularly for the higher-molecular-weight compounds, which required more collisions to reduce the kinetic energy of the ions for trapping than a lower- molecular-weight compound such as arginine. On the other hand, higher pressure only increased the multi- plier noise with no enhancement of the signal. Ions were gated into the trap over a 1-1000 ms time range. The signal level increased from 1 ms to a maximum signal level at 70 ms for the tuning solution containing arginine (200 ng/yL) and gramicidin S (100 ng/yL). Beyond 70 ms the signal level decreased along with the mass resolution due to space charging. This decrease was also noted at shorter ionization times for more concentrated solutions. The q2 for ion injection was dependent on the mass of the ions being analyzed. The optimal q2 value for trapping ions decreased with mole- cular weight. Arginine (MW 174) has an optimal q2 value of 0.2, while gramicidin S (MW 1141) optimized at a q2 value near 0.15 and P-endorphin (MW 3463) optimized around 0.13-0.14. Changing the qz value by a factor of two (e.g., from 0.15 to 0.3) could result in a loss of 90% of the signal level. This situation would

limit the ability to detect a broad molecular weight distribution. However, the measurement of the neuro- peptide could be accomplished under near optimal conditions with a fixed ql near 0.14 since the molecular weight range varied less than a factor of three. (The optimal q2 value was selected as the mean between the qz of the lowest and highest molecular weight neuro- peptide that would be determined). Finally, ions in the trap needed little time for additional desolvation prior to mass analysis. Holding the ions for up to 250 ms only resulted in a 5 1 0 % increase in signal level. This was not unexpected in the electrospray system used in this research, since the desolvation of the ions occurs before they are gated into the ion trap. This is not the case in other electrospray-ITMS designs.14-16

Flow injection of neuropeptide stannards a-MSH, a-endorphin, and P-endorphin solutions were introduced to the electospray interface individually (100 pmol/yL, 0.5 pL injected) through flow injection. The mass spectra obtained showed several multiply charged ions (Fig. 1) allowing the calculation of mol- ecular weight. Since electrospray is a soft ionization technique, no fragmentation was detected. However, the mass spectra acquired on the ITMS show slightly less charging compared to the spectra obtained from the quadrupole system. It is feasible that collisional damping of ions for storage in the trap and ion motions

(a) 556 [M+3HI3+

833 [M+ZH]*+

50

1666 [M+H]+

0 300 600 900 1200 1500 1800 mlz

Figure 1. Electrospray-ITMS mass spectra of various neuropeptides for a flow injection of 100 pmol/pL with 0.5 pL injected. (a) a-MSH (MW 1665), (b) a-endorphin (MW 1746), (c) /3-endorphin (MW 3463).

336 PERFUSION HPLC/ELECTROSPRAY-ITMS OF NEUROPEPTIDES

0

generated by the trapping RF fields (qz value) result in charge stripping increasing the m/r values.

0 o r m a m

HPLC/electrospray-ITMS A mixture of five neuropeptides-ACTH, a-MSH, a-endorphin, P-endorphin, and dynorphin B spiked into water (10-30 pmol/yL, 0.5 yL injected)-was used to test the feasibility of on-line capillary HPLC/electrospray-ITMS for the separation and detection of neuropeptides. With our optimized HPLC gradients, the separation took about 1 h to finish. The identification of neuropeptides in the acquired full-scan spectra was achieved by comparing the measured mol- ecular weight and retention time with the molecular weight and retention time of a standard. Obviously, the long separation time was not desirable; however, further reduction of the separation time by increasing the initial organic content in the mobile phase as well as by increasing the gradient slope ended up with co- eluting peaks for several of the neuropeptides. Therefore, perfusion chromatography was investigated to greatly reduce the analysis times.

Capillary perfusion HPLC lelectrospray-ITMS The coupling of perfusion chromatography with elec- trospray posed some minor problems due to flow-rate incompatibilities. In order to maintain the advantages of fast separation and minimize peak broadening, the perfusion column needs to be coupled to the electro- spray spray chamber directly. However, significant de- crease in ion current was observed when the electro- spray was operated at 30-40 yL/min (the flow rate of the perfusion column) due to difficulties in nebulization and in desolvation. While a post-column split could be incorporated, the dead volumes of the splitter and electrospray needle assembly could significantly reduce the resolution and speed of the perfusion separation.

A modification to the spray chamber was made to enable the successful direct coupling of the perfusion column to the interface by placing a stainless steel cap with a 6mm orifice in the center of the nitrogen counterflow tube. The center of the opening in the cap aligned with the gold plated electrospray capillary ori- fice. The cap extended about 2 mm beyond the end of the entrance glass capillary, and was held at a potential of -3200 V (end-plate potential). This capillary shield converges the nitrogen counterflow gas in front of the capillary entrance orifice, providing better heating and drying to desolvate the droplets formed by electrospray before they reach the capillary entrance. The capillary shield initially described by Whitehouse and co-workers' is similar to the liquid shield reported by Henion and co -worke r~ ,~~ enabling higher flow-rate operation, although flow rates beyond 100 yL/min have not been evaluated with the capillary shield (whereas the liquid shield has been reported to operate up to 2 ~ n L / m i n ~ ~ using another electrospray interface). Figure 2 shows that this modification maintains elec- trospray response within a factor of four, from 1 yL/ min to 60 yL/min for a constant level of gramicidin S infused into the system. Without this shield, electro- spray response decreased by a factor of 10 when the flow rate was increased from 2 yL/min to 10 yllmin. The electrospray mass spectra recorded at the higher

100

.

flow rates were similar to those at a lower flow rate, with no additional noise or signals from solvated ion or liquid droplets.

The perfusion HPLC/electrospray ITMS total-ion current chromatogram for 10 pmol/yL (0.5 yL injected) of dynorphin A standard (Fig. 3) shows the advantages of perfusion chromatography. The analysis is completed in only about 3min and dynorphin A showed a peak width at half height of 7 s (about 3600 theoretical plates). The mass spectrum of dynorphin A exhibits the multiply charged ions for the addition of 2-5 protons to the peptide (Fig. 3(b)).

On-line perfusion capillary HPLC/electrospray- ITMS combination could detect subpicomole levels of neuropeptides spiked into a canine serum extract. Figure 4 shows the total ion current chromatogram for the serum extract spiked with a-MSH, a-endorphin, and P-endorphin using the scan mode on the ITMS (mlz 50-650 in about 200 ms using resonance ejection to extend the mass range by a factor of three). The peaks for these three neuropeptides could be detected in less than 5min for subpicomole quantities. The corresponding signals could be detected with a higher signal-to-noise ratio in the extracted ion chromato- grams, displaying mlz 1747 for a-endorphin, 1734 for P-endorphin and 556 for a-MSH. The analysis of blank

0 1 : : , - '. J .5 1 1.5 2 2.5 3 3.5 4 4.5 5

Time (min)

100- lhl 1 1-1

[M+3HIG I 535

al 802 - a J . . . .

600 7M) 800 900 m.?

Figure 3. Perfusion capillary HPLClelectrospray-ITS (a) total ion current chromatogram of 10 pmol (20 pmol/pL 0.5 pL injection) of a solvent standard dynorphin A 1-13 and (b) mass spectrum of dynor- phin A 1-13.

PERFUSION HPLC/ELECTROSPRAY-ITMS OF NEUROPEPTIDES 337

100

.- 22 $ 50

._ !2

.- -

.- - U

Figure 4.

0.5 pmol 0.3 pmol 1 pmol a-Endorphin PEndorphin a-MSH \ I

I 1 1 I’ I’

I , I . , I I I I I I 1 I 1

1:15 2:31 4:OO 5 3 2 7:03

Time (min)

Perfusion c a d a r v HPLClelectrosmav-ITMS total ion cur- . a . <

rent chromatogram for a canine serum extract spiked with three neuropeptides, 0.5 pmol (1 pmollyL 0.5 yL injected) of a-endorphin (retention time 3.3 min), 0.3 pmol (0.6 pmollyL, 0.5 yL injection) of j3-endorphin (retention time 3.6 min) and 1 pmol (2 pmollyL, 0.5 yL injected) of a-MSH (retention time 4.5 min).

serum (not shown) demonstrated that these three peaks were unique to the spiked serum sample. The scan speed of the ITMS (200 ms/cycle or 300 ms/cycle using a 100 ms desolvation time) was sufficient to character- ize these rapidly eluting peaks (4-5 s peak width at half height) without introducing distortion. The perfusion column exhibited a slightly lower number of theoretical plates (-12 000) compared to the capillary C18 analysis previously described, but offered a different selectivity increasing the resolution between adjacent neuropep- tide peaks.

While perfusion chromatography offers rapid elution times and better peak resolution compared to capillary LC, capillary LC/electrospray-ITMS sensitivity (signal/ noise) was 2-3 times better than with perfusion LC. This slight loss in sensitivity is attributed to lower peak concentration for the target neuropeptide and operat- ing at non-optimal flow rate for electrospray (35 pL/ min instead of 2-5 pLlmin). Also, potential problems in loss of resolution due to biological material entering the pores packing material could be handled through an in-line 0.2 pm filter before the column or by ultrafil- tration of the sample. Using an in-line filter frit before the column enabled the analysis of numerous serum sample extracts ( > 20) without loss of chromatographic resolution or change in peptide retention.

CONCLUSION Perfusion chromatography coupled to electrospray- ITMS enabled separation and subpicomole detection of target neuropeptides (1000-5000 molecular weight range) in a serum extract. The use of perfusion chroma- tography enabled the analysis of 8-10 samples per hour compared to the 1 or 2 analyses that would be per- formed using capillary chromatography. The combi- nation of electrospray with an ITMS provided sensitivi- ties of detection of these components eluting from the perfusion column at subpicomole levels under full-scan acquisition conditions. While the commercial coupling of electrospray and the ITMS is not available, such combinations have been demonstrated to achieve the goal of increasing sensitivity relative to a mass analyzer with a lower ion-sampling cycle. However, the electrospray-ITMS coupling requires the optimization of parameters, mostly on the ITMS to realize this

increased sensitivity. For the analysis of the neuro- peptides discussed, it was found that pressure (-4 x Torr), ionization time (70 ms) and qz for trapping (0.14) were critical and required optimization to achieve the optimal sensitivity.

Acknowledgement This research was supported by NIDA Grant No. 5-R01 DA06315.

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