Anal. Chem. 1994,66, 2103-2109
Quantitative Capillary Electrophoresis- Ion Spray Mass Spectrometry on a Benchtop Ion Trap for the Determination of Isoquinoline Alkaloids Jack D. Henion,' Alex V. Mordehai, and Jianyi Cai Analytical Toxicology Dkgnostic Laboratory, College of Veterinary Medicine at Cornell, Cornell University, 927 Warren Drive, Ithaca, New York 14850
The quantitative determination of some isoquinolioe alkaloid natural products is described using combined capillary electrophoresis-ion trap mass spectrometry (CE/MS). The benchtop ion trap is a previously describedmodifiedcommercial VarianSaturn I1 system equipped with M in-house constructed atmospheric pressure ionzation (API) ion source which was operated in the ion spray CE/MS mode. All relevant operational parameters have been optimized for the CE/MS experiments described in this work. A coaxial sheath flow of solvent buffer was usedto facilitate ion spray CE/MS operation. Analysis of synthetic mixtures containing nine related isoquinoline alkaloids provided full-scan mass spectra for these compounds with injected quantities as low as 370 attomole. Thesignal-to-noiseratio wasbetter than 10/1 fortheextracted ion current electropherograms of the parent ions for these compounds with injected quantities in the 370-510 attomole range. CE/MS analysisof methanolextracts from the natural bark of Pbellodemiron wilsoniias well as an herbal medication provided full-scan mass spectra for the identificationof major and minor components in the mixture. Quantitative analyses were carried out using tetrahydroberberine as an internal standard. The standard curve ranged from injected levels of 0.6 to 16 pg (1.7 to 45 fmol) and had correlation coefficients of 0.998 and 0.999 for berberine and palmatine, respectively. These results suggest that the ion trap CE/MS system is sufficiently sensitive and reliable to provide acceptable quantitative analysis of synthetic and natural product mixtures. High-performance capillary electrophoresis (HPCE) is recognized as a powerful technique for the separation and analysis of samples composed of charged species which range from small ions to large The coupling of capillary electrophoresis with mass spectrometry (CE/MS) offers added capability by providing important information necessary for the identification and confirmationof components in complex mixtures. On-line CE/MS was first reported by Smith et al.4 and later by Lee et al.5 where capillary electrophoresis was coupled to a quadrupole mass spectrometer using an atmospheric pressure ionization electrospray and an ion spray interface, respectively. Since then, other types of mass (1) Karger, B. L.; Cohen, A. S.;Guttman, A.J. Chromatogr. 1989,492,585-614. (2) Wallingford, R. A.; Ewing, A. G. Ado. Chromatogr. 1989, 29, 1-76. (3) Huang, X.;Luckey, J. A.; Gordon, M. J.; a r e , R. N.AMI. Chem. 1989,61, 766-770. (4) Olivares, J. A.;Nguyen,N.T.;Yonker,C.R.;Smith,R. D. AMI. Chem. 1987, 59, 1232-1236. ( 5 ) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. J. Chromatogr. 1988,458, 3 13-321.
QQQ3-27QQ/94/Q38&2 7Q35Q4.5QIQ Q 1994 American Chemlcal Society
spectrometers have been employed.61~ A recent review highlights developments in the field.* One potential limitation with CE is the limited sample volume used due to the small inside diameter of the capillaries. Sample injection volumes are normally confined to the low nanoliter and low picomole range in order to maintain high separation efficiency. Although detection of subfemtomole levels of proteins using full-scan on-line CE/MS has been reported? the concentration detection limit for CE/MS is often unsatisfactory. One approach to improve the sensitivity of CE/MS is to increase sample loading using capillaries with larger inner diameters. lo Alternatively, sample preconcentration techniques may be used,l1J2 Other approaches include varying thevoltagegradient, which reduces the elution speed,l0 or using selected ion monitoring (SIM) techniques13 during CE/MS experiments. Since the above approaches frequently either are nonuniversa1 or require prior knowledge of the sample components, a more universal technique is needed. Recent exciting developments which implement electrospray ionization on an ion trap mass spectrometer appear to offer some new analytical potential for mass spectrometry experiment^.^^ Related efforts reported by our group15J6as well as othersl7J8abhave suggested great potential for the combination of CE with an ion trap mass spectrometer equipped with electrospray. (6) Mosely, M. A.; Detering, L. J.; Tomer, K. B.; Jorgenson,J. W. Rapfd Commun. Mass Spectrom. 1989, 3, 81-96. (7) Hallen, R. W.; Shumate, C. B.; Siems, W. F.; Tsuda, T.; Hill, H. H., Jr. J. Chromatogr. 1989,480, 233-245. (8) Smith, R. D.; Wahl, J. H.;Goodlett, D. R. AMI. Chem. 1993,65,574A-S84A. (9) Wahl, J. H.; Goodlett, D. H.; Udscth, H. R.; Smith, R. D. A M / . Chem. 1992, 64, 31943196. (10) Altria, K. D. LC-GC 1993, Il(6), 438-442. (11) Cai, J.; El Rassi, Z . J. Uq.Chromatogr. 1992, I5 (6&7), 1179-1192. (12) T h o m p n , T. J.; Foret, F.; Vouros, P.; Karger, B. L. AMI. Chem. 1993,65, 9Qo-906. (13) Johansson, I. M.; Pavelka, R.; Henion, J. D. J. Chromclrogr. 1991,559,515528. (14) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Chem. Soc. 1990, 112, 5668-5670. (15) Henion, J. D.; Mordehai, A. V.; Wachs, T.; Huggins, T. G.; Cai, J. Rccent Developmentsin CE/MS Determination of Pharmaceutical and Environmental Compounds. Prescntcd at The Fifth International Symposium on Capillary Electrophoresis (HPCE93). Orlando, FL, January 25-28, 1993. (16) Henion, J. D.; Mordehai, A. V.; Lim, H. K.; Cai, J. Fundamentals and R e n t Applications on an Atmospheric Pressure Ionization-Differentially Pumped Benchtop Ion Trap. Prcsenttd at The 41st ASMS Conference on Mass Spectrometry and Allien Topics, San Francisco, CA, May 31-June 4, 1993. (17) Schwartz, J. C.; Jardine, I. Capillary Electrophoresis Ion Trap Mass Spectrometry. Prcsentcdat The40thASMSConfermceonMassSpectrometry and Allien Topics, Washington, DC, May 31-June 5, 1992, pp 707-708. (18) (a) Ramsey, R. S.; Asano, K. G.; Hart, K. J.; Gocringer, D. E.; Van Berkel, G. J.; Mcluckey, S. A. Combining Capillary Electrophoresiswith Electrospray Ionization Trap Mass Spectrometry for Biopolymer Analysis. Rcsentcd at The 41st ASMS Conference on Mass Spectrometry and Allien Topics, San Francisco, CA, May 31-June 4, 1993, pp 749a-749b. (b) Ramsey. R. S.; Gocringer, D. E.;Mcluckey, S . A. Anal. Chem. 1993, 65, 3521-3524.
Ana&tical Chemistry, Vot. 66, No. 13, Ju& 1, 1994 2103
We have selected a series of isoquinoline alkaloids to determine the feasibility of conducting quantitative CE/MS analysis experiments using the benchtop ion trap mass spectrometer. Since it has been shown that the pharmacological activities of many folk medicines are due in part to these quaternary ammonium compounds, we have used the described benchtop ion trap system to carry out quantiative analyses for two isoquinoline alkaloids present in a Phellodendron wilsonii bark extract. Since each individual alkaloid has its own medical use, it is important to develop a technique suitable for the separation as well as qualitative and quantitative determinations of isoquinoline alkaloidsin natural products in order to better utilize these resources. Most published analyses of alkaloid samples have involved high-performance liquid chromatography (HPLC) and thinlayer chromatography (TLC).19*20Due to the basic nature of the alkaloids, chromatographic peak tailing and unsatisfactory resolution are often observed with HPLC separations. Although some quaternary amine additives may be used to improve the chromatographic peak shape, they are frequently undesirable for on-line LC/MS analyses. Capillary electrophoresis techniques, however, can provide an alternative, improved separation mode for characterizing samples containing this class of compounds. P. wilsonii is one of the important plants that contains a number of pharmacologically interesting alkaloids. The dried tree barks of several P. wilsonii species are traditionally used as medicine in China and other countries for the treatment of dysentery and jaundice.21 Several alkaloids have been isolated, and their antimicrobial activitieshave beenreported.22 In this paper, we describe the separation, identification, and quantitation of selected isoquinoline alkaloids present in the extracts of the bark of P. wilsonii and an herbal medicine tablet using capillary electrophoresis-ion spray-ion trapmass spectrometry on a modified benchtop ion trap with an inhouse-constructed liquidshield atmospheric pressureionization interfa~e.~~?~~
EXPERIMENTAL SECTION Reagentsand Materials. Berberine chloride and palmatine chloride (hydrate) used for quantitation were purchased from thesigma Chemical Co. (St. Louis, MO). The Emerson tablet and the extract of the bark from P. wilsonii, as well as the other isoquinoline alkaloid standards used in the this study, were kindly supplied by Dr. Wu-Nan Wu of the R. W. Johnson Pharmaceutical Research Institute. The chemical structures and molecular weights of the isoquinolinealkaloid compounds used in this study are shown in Figure 1. Organic solvents, HPLC grade water, and all the other chemicals used for capillary electrophoresis were analytical-reagent or reagent grade and were obtained from Fisher Scientific (Rochester, NY). All the electrolytes were prepared daily and filtered through 0.2-pm Nylon HPLC syringe filters (Krackler Scientific, Albany, NY) before use. Uncoated fused-silica (19) Bugatti. C.; Colombo, M. L.; Tome, F. J. Chromutogr. 1987, 393, 312-316. (20) D?ido, T.J. Chromurogr. 1988, 439, 257-266. (21) LI,C. P. Chinese Her601 Medicine; U.S.Department of Health, Education, and Welfare, Public Health Service, National Institute of Health, 1974. (22) Wu, W-N.; Mitsher, L. A.; Beal, J. L. LLOYDIA, 1976, 39 (4), 249-252. (23) Mordehai, A. V.; Hopfgartner, G.; Huggins, T. G.; Henion, J. D. Rupid Commun. Muss Spectrom. 1992, 6, 508-516. (24) Mordehai, A. V.; Henion, J. D. Rupid Commun. Muss Spcctrom. 1993, 7 , 205-209.
2104
Am@alChemh&~, Vol. 66,No. 13, July 1, 1994
Jatronhizlne
Berberlne
Mw=338
we336
Thalphenlne MW.352
ocy
oat,
Phellodendrlne
c%
MW.342
=Y OH
Columbamlne MWe338 MW.355
ocy
9
Palmatine
=HI
Magnoflorlne
MWd52
=HI
MW.342
Y
OCH,
0%
Figure 1. Structures, names, and molecular weights of lsoquinoline alkaloid standards.
capillaries with dimensions of 2 5 , 50-, and 100-pm i.d. and 150-1 90-pm 0.d. were purchased from Polymicro Technologies (Phoenix, AZ). Capillary Electrophoresis/MassSpectrometry. A Beckman Model P/ACE 2050 system (Palo Alto, CA) used for this study was kindly supplied by Beckman Instruments, Inc. (Palo Alto, CA). Thecommercial instrument was slightly modified in that both the safety lock between the capillary ends and the temperature controlling system were bypassed. Sample analysis was performed on a capillary having a total length of 90 cm with the first 25 cm housed in a modified cartridge holder13with the remaining 65 cm extended through ambient air to the ion spray CE/MS interface. The latter served as the cathode end of the CE capillary and consisted of a coaxial capillary arrangement of three concentric capillaries (Figure 2, vide infra). The ion spray interface used in this study was constructed in-house. Some modifications have been made to the ion spray interface described earlier.2s The first stainless steel dead volume tee, which serves to deliver the coaxial sheath flow liquid, was replaced by a PEEK tee and finger-tight fittings (Upchurch Scientific Inc., Oak Harbor, WA). Figure 2 shows the details of the modified interface. PEEK tubing was used not only as a seal around the capillaries but also as a guide between the sheath flow tube and CE capillary (see Figure 2). This modification enables the easy exchange and adjustment of the CE capillary without disturbing the position of the ion spray interface. The CE capillary was housed inside a stainless steel sheath capillary (254-pm i.d. X 508-pm o.d.), through which the make-up buffer or solvent flow was introduced. A larger stainless steel capillary (584-pm i.d. X 902-pm 0.d.) concentric to the inner capillaries served as the (25) Huggins, T.G.; Henion, J. D. Electrophoresis 1993, 14, 531-539.
jb
/'
b
.
0
0
c
a
h
I I I I I I I
Figure 2. Schematic illustratlon of the ion spray CE/MS interface with adjustable CE capillary: (a) CE capillary, (b) stainless-steelsheath flow capillary, (c) stainiess-steel nebulizing capillary, (d) sheath flow inlet, (e) nebulizing gas inlet (N2,50 psi), (f) PEEK tubing, (9) finger-tight fittings, (h) PEEK tee, (i) stainless-steel tee, (j) high-vottage power supply connection (3-5 kV).
nebulizing gas tube through which dry nitrogen gas was introduced at 50 psi. The sheath flow liquid used was 2 or 5 mM ammonium formate in 100%methanol and was delivered at 10 pL/min by a Model 140A solvent delivery system (Applied Biosystems, Inc., Foster City, CA). On-line UV detection was performed at 254 nm with the detector located 20 cm from the anode end of the CE capillary. Difficulties can occur with optimum positioning of the capillary window within the UV detector for stable and sensitive online UV detection. This may be overcome by the use of two pieces of a larger fused-silica capillary (250-pm i.d. X 350pm 0.d.) tubing which serves as a guide at the inlet and outlet of the CE capillarydetection window. With this modification, on-line UV detection and analytical ruggedness may be significantly improved. The capillary electrophoretic separation was carried out by applying 30 kV to the anode end while 3 kV was applied to the cathode via the ion spray interface high-voltage supply, resulting in a potential difference across the CE capillary of 27 kV. Sample injection was made at the anode end in the electrokinetic mode for 10 s during which a voltage of 10 kV was applied at the anodeend and 3 kV at the ion spray interface, which resulted in a 7-kV net voltage drop across the CE capillary. Because the nebulizing gas was found to cause siphoning and laminar flow inside the CE capillary (vide infra), it was turned off during injections to avoid this problem and the possible introduction of air. Determination of Injection Quantity. The injection amount, Q, was determined by the total mobility of the analyte during injection (when the nebulizing gas is turned off') according to the following relationship:
where r is the inner radius of the capillary, C is the concentration of the solute, I is the length of the capillary from inlet end to the detection point, and tinj and tl are the duration of injection and the time required for the analyte to migrate a distance I, respectively. Mass Spectrometry. The ion trap mass spectrometer used in this study was the same as discribed earlier.24 The in-
house modified benchtop ion trap mass spectrometer was equipped with a differential pumping system and atmospheric pressure ionization interface.23 The helium pressure in the ion trap region was maintained at 6 X l t 3 Torr (corrected). In all the CE/MS experiments, the scan range was from m / z 150 to 400. Extraction and Sample Preparation. Five grams of the dry bark from P. wilsonii was crushed and extracted twice with 50 mL of methanol. The extract was filtered and evaporated to dryness. The resulting 0.42 g of dark brown residue was reconstituted with 42 mL of methanol and stored at -20 OC. The methanolic solution was diluted with HPLCgrade water immediately before analysis by CE/MS. The herbal medicine tablet (0.2723 g) was crushed and extracted twice with 2 mL of methanol. The remaining solid residue was extracted with 4 mL of a water/methanol (1:1) solution. Following centrifugation, the supernatants were combined and evaporated under reduced pressure. The final residue was reconstituted in 0.5 mL of water/methanol (1:l). Isoquinoline alkaloid standards were individually kept in methanol (1 mg/mL). Synthetic mixtures were prepared by mixing the methanolic solutionsof the standards and diluting with HPLC-grade water to obtain the desired concentrations. All samples were filtered through 0.2-pm Nylon microcentrifuge filters (Lida Manufacturing Corp., Kenosha, WI) on a micocentrifuge (Tomy Seiko Co., Tokyo, Japan) to avoid capillary plugging. RESULTS AND DISCUSSION Effect of Operational Parameters. To optimize the sensitivity, separation efficiency, and resolution of the isoquinoline alkaloids, several operational parameters were studied. Composition of CE Buffer. The optimum separation conditions for the isoquinolinealkaloidswere carried out using running electrolytes of different pH (pH 2.5-6.0), organic content (methanol &SO%), and concentration (50-1 00 mM) of ammonium acetate. The presence of organic solvent in the running buffer serves to improve the sensitivity of detection for the analytes under ion spray ~0nditions.l~ The optimum separation was obtained with a running electrolyte containing 40% methanol and 60 mM ammonium acetate at pH 4.5. Although higher organic content could improve the resolution by reducing the electroosmotic flow, it was found to significantly increase the separation time and exhibit poor electrophoretic peak shape. Nebulizing Gas. Nebulizing gas is critical for good sensitivity for the ion trap mass spectrometer equipped with the liquid shield interface.23 The effect of nebulizing gas pressure on the mass spectrometric sensitivity is shown in Figure 3A with all other conditions held constant. A CE capillary of 50-pm i.d. and 90-cm total length was used with 30 kV at the anode and 3 kV at the cathode (ion spray interface). Injection was performed electrokinetically for 10 s with 10 kV at the anode and 3 kV at the cathode. A 5 ng/pL (51 fmol) amount of berberine was loaded on-column. The CE buffer was 60 mM ammonium acetate, pH 4.5, in 40% methanol. The sheath flow liquid was 5 mM ammonium formate in methanol and was delivered at 10 pL/min. The ion trap was operated under low "up-front" CID conditions AnawicalChemistry, Vol. 66, No. 13, July 1, 1994
2105
E 402
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.-C
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8
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25
35
45
55
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0
25
50
100
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Flgure 3. Effect of ion spray nebulizinggas on CE/MS signal-to-noise ratio, intensity, electroosmotic flow, separation efficiency, and mass spectrometric sensitivity. (A) Effect of ion spray nebulizing gas on signal-to-noise ratio and intensity. CE conditions: CE capillary had a 50-pm i.d. and 90cm total length; CE buffer was 60 mM ammonium acetate, pH 4.5, 40% methanol; separation was performed with 30 kV at the anode and 3 kV at the cathode (ion spray interface); a solution containing 5 ng/pL (51 fmol) of berberine was injected oncolumn, and the sheath flow was 5 mM ammoniumformate in methanol at a flow of 10 pLlmin. Ion trap mass spectrometer conditions: low “up-front” CID conditions (nozzle-skimmer potential = 52.4 V). (B) Effect of ion spray nebulizinggas on electroosmoticflow and separation efficiency (percent increase Inthe apparent electroosmotkflow, percent decrease in theoretical plate number per meter) as a function of CE capillary inner diameter. CE conditions: separation was performed with 20 kV at the anode and 3 kV at the cathode (ion spray interface); 1 ng/pL phenol was injected on-column. Nebulizing gas was turned off to measure the true electroosmotic flow, and the gas pressure was kept at 50 psi to determine its effect on the EOF; on-line UV detection was used at 254 nm positionedat 20 cm from the inlet end. Other CE conditions were the same as in part A.
(nozzle-skimmer potential = 52.4 V). Poor sensitivity is observed with low nebulizing gas pressure using the liquid shield interface;e.g. pure electrosprayconditions produce little or no ion current with this system. As is shown in Figure 3A, the peak intensity is increased more than 50-fold as the nebulizing gas pressure is increased from 20 to 50 psi. The optimum nebulizing gas pressure in terms of the signal-tonoise ratio was found to be 50 psi. While the mass spectrometricsensitivity is optimized,the ion spray nebulizing gas can also cause some undesirable hydrodynamic flow in the CE capillary as a result of the partial negative pressure created by the high linear velocity of the nebulizing gas at the capillary tip. In order to evaluate the effect of the nebulizing gas on the bulk liquid flow inside the CEcapillary,we measured the percent increase in apparent electroosmotic flow (EOF) and the percent decrease in separation efficiency (theoretical plate number per meter, N/m) as a result of the nebulizing gas flow. While the other experimental conditions were kept unchanged, capillaries of different inner diameters were evaluated. Phenol (1 ng/pL) was used as a marker to measure the EOF. The true EOF must be measured in the absence of nebulizing gas, which makes it impossible for a mass spectrometer equipped with the liquid shield interface to operate as a detector since there is no signal from the mass spectrometer without the nebulizing gas. To overcome this, on-line U V detection was used (254 nm with the U V window positioned 20 cm from the inlet end) in this study. The results showed that when capillaries of 50 pm and smaller inner diameter were used, neither electroosmoticflow nor separation efficiency were altered significantlyby the nebulizing gas flow (see Figure 3B). Although less hydrodynamic flow was 2106
AnalyticalChemkby, Vol. 66, No. 13, July 1, 1994
observed in capillaries of smaller inner diameter, the capillary plugging that may be experienced with these capillaries dictated that 50-pm-i.d. capillaries be used for most of the work in this study. It should be noted, however, that, in those instances where pure electrospray (no nebulizing gas) is used during CE/MS experiments using alternative API interfam,4J3J8the problem of induced EOFdescribed above should not be a problem. Some researchers have attempted to eliminate hydrodynamic flow by lowering the level of the anode reservoir to compensate the pressure drop across the CE capillary due to the nebulizinggas flow.1° However, this approach is somewhat complicated because the necessary height difference between inlet and outlet ends of the CE capillary to achieve zero hydrodynamic flow was found to be sensitive to temperature, electrolyte composition, and particularly the organic content in the buffer electrolyte. In this study the outlet end of the CE capillary, which was the tip of the ion spray interface tip, was maintained at the same height as the inlet end. To avoid the introduction of air at the inlet end during vial switching, it is beneficial to turn off the nebulizing gas while injection is made. Other Parameters. It was also found that the condition of the CE capillary inner surface plays an important role in the separation of the title basic compounds. Due to the basic nature of these quaternary amines, peak tailing has been observed with uncoated CE capillaries. However, peak shape may be improved when the capillary has been pretreated. This involves a rinse of the capillary with methanol, water, and 0.1 M sodium hydroxide, as well as an organic-aqueous solution of ammonium hydroxide (0.1 M, 20% acetonitrile) followed by 100% acetonitrile. The capillary provides the best performance when it is kept in methanol while not in use. These experiments also revealed that the best sensitivity in terms of signal-to-noise ratio was obtained when the CE capillary exit tip was flush relative to the sheath flow capillary at the ion spray interface tip (see Sprayer Tip; Figure 2). Three different sheath flow compositions were studied including formic acid, ammonium formate, and ammonium acetate (2-5 mM) in methanol. The CE/MS response to the title compounds was independent of the above sheath solutions, but it was affected by its flow rate. The optimum sheath flow for the present system was found to be 10 pL/min. The effect of the sheath flow was primarily upon the background noise and the overall sensitvity of the analyte. Analysis of a Synthetic Mixture. Figure 4 shows the fullscan total ion current electropherogramobtained for a synthetic mixture containing low femtomole quantities injected into a 50-pm-i.d. capillary. Seven of the eight isoquinoline alkaloids are shown separated. Columbamine and palmatine (peaks 3 and 4) co-eluted under these CE/MS conditions. “Up-front” collision-induced dissociation (CID) was used to obtain fragmentation information for each component. This was done by the application of a potential difference across the first vacuum which leads to the acceleration of the ions and their collision with neutral molecules in this highpressure region. This potential determines the collision energy. When this CID energy is sufficiently high, sample ions will dissociate into fragment ions. In this study the potential difference was set at 70 V to induce fragmentation, and 52.4
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Fbure 4. Total bn current electropherogram of a Synthetic mixture contalnhg known koquinolinealkaldds. CE conditionssame as In Flgure 3A. Iontrap mass m c o n d l t k n s : high “upfront” CID(nozzbskimmer potentiel = 70 V). Peak assignment and amount Injected on-cdwrm: (1) behedne, 51 fmol, (2) thalphenlne, 48 fmol, (3) dumbamhe, 46 fmol, (4) palmatine, 44 fmol, (5) jetronhlzine, 46 fmol, (6) phelbdendrlne,43 fmol, (7) tetrahydropalmatine, 40 fmol, (8) magnoflorlne, 37 fmol. 6
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Flgurr 5. On-llm CE/MS background-substracted mass spectra of b”obtalned wlth the ion trap massspectrometer operatedunder (A) low “up-front” CID conditions (nozzle-skhmer potentlal = 52.4 V), and (8) hlgh “up-front” CID conditbns (nozzle-sklmmer potentiel = 70 V). The quantky of berberlne keded Into the capillary for these mass spectra was 51 fmol. other experimental conditions are the same as In Flgwe 4.
V when no CID was desired. When low “up-front”CID energy was used, the predominant ion obtained was the parent quaternary ion, as is shown in Figure SA. In these instances the SIN is comparable in TIC electropherograms obtained under both low and high “up-front” CID conditions (data not shown here). However, there is often an apparent improve-
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Figure 0. Total Ion current electropherogram of the extracts of herbal mediche tablet. See Table 1lfor peak assignments. Experimental conditions are the same as in Figure 4.
ment in sensitivity for the analyte due presumably to improved focusing of ions under the higher CID conditions. Figure 5B shows the background-subtracted “up-front” CID mass spectrum of berberine obtained by on-line CE/MS using high “up-front” CID energy with the synthetic mixture containing low femtomol quantities of each compound. This CID mass spectrum is very similar to the full-scan CID mass spectrum obtained under tandem mass spectrometry (MS/MS) conditions using our Sciex TAGA 6000E tandem triple quadrupole mass spectrometr (data not shown). The fragmentation observed using “up-front” CID is sensitive to the temperature of the API interface.23 This temperature is controlled by a heater attached to the liquid shield. It was found that with the same potential difference in the first vacuum region a higher temperature on the heated liquid shield would cause more fragmentation. One explanation for this phenomenon is that a higher temperature will lead to better declustering and desolvation of the sample ions in the atmospheric pressure region which will facilitate the collision-induced dissociation process. Nevertheless, with proper control of the “up-front” CID conditions reproducible “up-front” CID mass spectra may be obtained. Identification of Components in Extracts of Bark and an Herbal Tablet. Since reproducible, structurally informative CE/MS full-scan background-substracted “up-front” CID mass spectra were obtained in this work, the components present could be identified in real-world samples, namely the extracts of P . wilsonii and an herbal tablet. The same experimental conditions as described above for the synthetic mixture were used. Figure 6 shows the total ion current electropherogramfor the herbal tablet extract whereas Figure 7 shows the corresponding data for the P. wilsonii bark. The identification of each peak was based on a comparison of the relative migration time and the background-subtracted ”upfront”CID mass spectrum from each component in the extracts with that of the standard under the same high “up-front” CID CE/MS conditions. Figure 8A-C shows the mass spectra for authentic phellodendine as well as this compound in each of the natural product extracts. The component identification is accomplished by comparison of the corresponding mass spectra as well as the relative migration times of each compound. These data are summarized in Table 1. Relative migration times rather than absolute migration AmWcaIChemktr~,Vd. 66, No. 13, July 1, 1994
2107
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T a M 1. Identltioation ot Compound8 In the Natural Product
Samples sample synthetic mixture
1
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1.033 1.134 0.870 0.890 0.902 0.937 0.955 0.971 0.983 1 .Ooo
1.135 0.893 0.936 0.969 0.983 0.983 1 .Ooo
1.033 1.135
identity berberine thalphenine columbamine palmatine jatrorrhizine phellodendrine tetra hydropalmatine magnoflorine unknown berberine unknown unknown unknown palmatine unknown phcllodendrine magnoflorine berberine unknown palmatine jatrorrhizine unknown phellodendrine tetrahydropalmatine magnoflorine
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4m a t t m l s
PHELMD~DRIHE,438 att-ls
times were used since this minimizes the effects from fluctuation in the CE bulk flow rate as a result of subtle roomtemperaturevariation and thechangein capillary inner surface conditions. Limit of Detection for Isoquinoline Alkaloids. In order to evaluate the sensitivity of CE/MS in terms of concentration and absolute amounts injected, results from capillaries of 25and 50-pm i.d. were compared. Several dilutions of the synthetic mixture were analyzed under low "up-front" CID conditions. Figure 9 shows the extracted ion current electropherograms for m / z 336, 352, 338, 356, and 342 for the synthetic mixture using a 25-pm i.d. capillary with 370-510 amol of sample injected on-column. Not surprisingly, the smaller i.d. capillary provides a better signal-to-noise ratio than one with larger (i.d. = 50 pm) when the same quantity of material is injected (data not shown). This is in agreement with results presented by Smith et a1.* Under optimum conditions, a full-scan CE/MS limit of detection (signal-tonoise ratio = 3) as low as 90 amol injected on-column may be achieved with, for example, magnoflorine (not shown). The other isoquinoline alkaloids studied in this work demonstrated limits of detection in the range from 90 to 130 amol of injected material. QuantitativeAnalysis of theIsoquinolineAlkaloids. In order to demonstrate the capability of capillary electrophoresis-ion 2108
AmlyticaiChemislry, Voi. 66,No. 13, Ju& 1, 1994
W F L O R I W E , 378 a t t m l c
342 1 ' " " " " 1 " " ' ' ' " / ' ~ " ' " ~ ' 1 ' " ~ ' " '
458 15:W
588
16:48
558 18:28
688 28:W
-
658 21:48
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swyl
Figure9. Exlractedioncwentelectropherograms of a synthetic mixtve of isoqubbakakkls. Iontrapconditbns: low"upfront"CID(noulesklmmer potential = 52.4 V). Compounds and the amounts injected on-column: berberine, 510 amol; thalphenlne, 480 amol; palmatine, 440 amok columbamlne, 460 amok jatrorrhizine, 460 amol; tetrahydropalmatine, 400 amok phellodendrlne,430 amol; magnoflorine, 370 amol. Caplliary i.d. = 25 pm. Other experimental conditions are the same as In Figure 4.
spray-ion trap mass spectrometry for quantitative analysis, the quantitation of berberine and palmatine in the natural product extracts was investigated. Calibration curves were made for the two components with standard solutions of berberine and palmatine containing the internal standard, tetrahydroberberine, maintained at 1 ng/pL (3.41 pg or 9.6 fmol injected on-column) for each point on the standard curve. Tetrahydroberberinewas chosen as the internal standard since it is not found in the P.wifsonii bark or herbal tablet extracts, and its structure is closely related to the compounds present in the natural products (Figure 1). An isotopically labeled internal standard was not available. The standard curves for berberine and palmatine were linear with correlation coefficients of 0.998 and 0.999, respectively, in the range studied. This range was selected due to nonlinear
Table 2. Quantltatlve Determinatbn of Berbertna and Palmathe by CE/MS
sample
Phellodendron wilsonii bark herbal medicine tablet
8.0
4.0
0.0
12.0
16.0
Amount Injected (pg)
Figure 10. Quantitative determination of berberine and palmatine by CE/MS. Internal standard is tetrahydroberberlne. Ion trap condltlons: low "up-front" CID (nozzle-skimmer potential = 52.4 V). Other experimental conditions are the same as in Figure 5.
mTtt4E
peak area = 2%
I\ INTw1(AL STAWDIU(D
8
peak ama = l1
L,
(..
,
,
I\
!,
, ; 7 \, ,,, : . 488
13:28
458 15:M
,
500 16:4B
-;,
,',
,
. , ,J
sc(y(I
TIM(nl)o
Figure 11. Extracted ion current proflle for quantltative determination of berberine and palmatine by CE/MS. The mlz 338 ion current proflle for berberine (882 fg), palmatine at mlz 352 (822 fg), and tetrahydroberberine at mlz 354 (3.41 pg) were taken from the lowest point of the standard curve shown in Figure 10.
behavior when higher levels of the target compounds were loaded into the capillary. Figure 10 shows the calibration curves which ranged from 0.6 to 16 pg (or 1.7 to 45 fmol) of each analyte injected on-column. Each point on the standard curve represents the average of two CE/MS measurements determined for the ratio of the analyte parent ion current peak area relative to the parent ion current peak area for the internal standard. Figure 1 1 shows the extracted ion current profiles for the most abundant ion ( m / z 354) of tetrahydroberberine (3.41 pg injected on-column) and the lowest point on the standard curve (Figure 10) corresponding to 682 fg of berberine ( m / z 336) and 622 fg of palmatine ( m / z 352) injected on-column. Each point on the standard curve shown in Figure 10 is the mean of the area ratios from duplicate analyses at each level shown. The corresponding analyte ions were used for the quantitation of berberine ( m / z 336) and palmatine ( m / z 352) in the extracts of the bark and that of the herbal tablet. Dilution of the extracts was necessary to obtain relative peak areas within the range of the calibration curve. The quantities of berberine and palmatine in the extracts determined from these standard curves are summarized in Table 2. Berberine was found to be the major component in the extract of the bark of P. wilsonii,which is in agreement with published results.Z1 CONCLUSIONS The experimental conditionsfor the benchtop atmospheric pressure ionization ion trap system described in this work
bergerine (wg/mg) palmatine (wglmg) 13.95 dry bark 0.0179 tablet
0.234 dry bark 0.0225 tablet
have been optimized for high sensitivity CE/MS applications. A full-scan on-column CE/MS limit of detection in the low attomole range may be achieved for some of the title compounds under optimum conditions. The components in the ion current profile at this level cannot be observed in the TIC, but they can be readily observed in the corresponding extracted ion current profile. In this work the target compounds may be observed in the TIC when low femtomole (3-5 fmol) levels are loaded into the capillary (data not shown). The described system also provides full-scan mass spectra from less material than is usually required for selected ion monitoring (SIM) experiments from quandrupole mass spectrometer systems4*5*8.9JlJ5or magnetic sector6 systems. The ability to obtain full-scan mass spectra from very low levels of compoundsin complex mixtures provides the potential capability of detecting unknown compnents in these mixtures rather than requiring a knowledge of what ions to monitor during SIM experiments. If the benchtop ion spray-ion trap system described in this work were to become commercially available, it could in principle be less expensive to purchase than the research-grade systems described pre~iously.*~J8 This report also describes the first example of quantitative capillary electrophoresis-ion spray-ion trap mass spectrometry. We have demonstrated that this technique may be used for the trace-level identificationand quantitativedetermination of isoquinoline alkaloids in a natural product sample. Optimization of ion spray nebulizing gas flow was found to be important for optimal sensitivity with the described liquid shield interface; it has little effect on the bulk flow in CE capillaries whose internal diameters are less than 50 pm, although it will significantly affect the bulk flow for largerbore capillaries. The modified sprayer for the described CE/ MS interface combination was found to be convenient for CE/MS applications when exchange or adjustment of CE capillaries is involved. ACKNOWLEDGMENT We thank Beckman Instruments for the loan of the P/ACE 2050 capillary electrophoresis instrument and the Eastman Kodak Co. for partial financial support of this work. Dr. Wu-Nan Wu of the R. W. Johnson Pharmaceutical Research Institute is gratefully acknowledged for supplying the isoquinoline alkaloid standards and the natural product samples. J.C. and A.V.M. express their appreciation for the support generously provided from Dr. H. K. Lim, of Wyeth-Ayerst Research, and Dr. T. Wachs. Received for review January 3, 1994. Accepted April 11, 1994.@ Abstract published in Advance ACS Absrracts, May 15, 1994.
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