Analysis of Coumarins by Micro High-Performance Liquid Chromatography-Mass Spectrometry with a Particle Beam Interface Achille Cappiello, Giorgio Farniglini, and Filippo Mangani lstituto di 5cienze Chlmiche. Universita d i Urbino, Urbina, Italy Bruno Tirillini Istituto di Botanica e arlo Botanico, Universita di Urbin o, Urbino, Italy Coumarins are a large group of compounds that are naturally present in plant tissues and that exhibit a wide range of pharmacological properties. Analytical methods based on chromatographic techniques and conventional detectors are inadequate to accurately anal yze coumarins in complex matrices such as plant extracts. In this article a new method based on a modified particle beam liqu id chromatography-mass spectrometry interface is described. The method allows specific and accurate determination of several coumarins in biological ma trices. An application rega rding the anal ysis of 18 coumarins in the extract of Snrfrnium perfoliatum L. is also reported. (J Am Soc Mass Spectrom 1995, 6, 132-139) oumarins are a large family of substances that ionization ten [4, 5], and electron attachment [6] have are usually extracted from plants, where they been employed successfull y. Cpursue a distinct biological role: production of Traldi and co-workers [7-10] have investigated the coumarins appears to increa se when the plant becomes structures of furocoumarin isomers, which cannot be the object of external aggression. Most of the interest in distingui shed with conventional mass spectrometric these compounds arises from their pharmacological techniques. The y established a new approach in the properties and some toxicological risks to humans. For investigation of these compounds based on high and example, scopoletin has a stabilizing effect on biolog i­ low energy collision-activated dissociation . cal membranes with a resultant hepatoprotective activ­ Some of the analytical methods described in the ity. Scopoletin also inhibits platelet aggregation and, literature rely on gas chromatographic-mass spectro­ moreover, it blocks the neurotransmitters of the cholin­ metric analyses of derivatized coumarins [11]. Meth­ ergic and adrenergic neuroeffector junctions. Pso­ ods that employ direct gas chromatographic injections larens, another group of cournarins, are characterized of untreated material also were proposed for selected by photobiological effects and are used in the pho­ compounds [12-14], Liquid chromatographic methods tochemotherapeutic treatment (PUVA therapy) of some are extensively used for the analysis of rather complex skin diseases with hyperproliferative conditions [1]. mixtures of different coumarins. These methods in­ Unfortunately, the pharmacological effectiveness of clude thin layer chromatography (TLC) [15] and high­ these drugs is impaired by side effects such as severe performance liquid chromatography (HPLC) methods dermatitis, erythema, phototoxicity, and possible in­ with reversed-phase and direct-phase separations [16, creased risk of sk in cancer, which is mainly related to 17]. TLC also is widely used for sample preparation the capacity of psolarens to form light-induced mono and clean-up procedures for plant extracts. Alcock et or bis adducts with DNA pyrimidine bases [2]. aJ. [18] described an interesting and pioneering appli­ Mass spectrometry has shown a great potential for cation of liquid chromatography-mass spectrometry structure elucidation and isomer characterization of analysis (LC/MS) of timbo powder extracts. They used several coumarins. Different ionization techniques like a glass-lined stainless steel microbore column coupled electron impact (En [3], pos itive and negative chemical to a mass spectrometer via a moving belt LC/MS interface. Current LC/MS interfaces allow realization of the inherent potential of both techniques, which thus ex­ Address reprint request s to Dr. Achi lle Cap piell o, Ist ituto di Scie nze Chimiche, Un iversita d i Urb ina, Piazza Rina scimento 6, 61029 Urbina, pands the application capabilities and greatly simpli­ Italy . fies the overall analytical procedures. In particular, the © 1995 American Society for Mass Spectrometry Received March 14, 1994 1044-0305/95/$9.50 Revised July 1, 1994 55011044-0305(94)00096-1 Accepted July 20, 1994 J Am Soc Mass Spectrom 1995,6,132-139 MICRO-HPLC/MS WITH A PARTICLE BEAM INTERFACE 133 particle beam (PB) interface [19-28] has proved valu­ ture was 40°C for all the experiments. The pressure able for LC/MS analysis of small molecules. It is fully was reduced to about 0.5 attn in the desolvation cham­ compatible with electron impact ionization (ED or ber, 0.3 torr in the second stage of the momentum chemical ionization (CD, with no significant constraints separator, and 5-8 X 10- 5 torr in the manifold of the regarding the liquid chromatography requirements or ion source. The ion source temperature was set at 250 analyte polarity, which makes it an invaluable tech­ °C and the analyzer was at 120 "C. The mass spectrom­ nique for analyses of complex mixtures. The analysis eter tuning and calibration were performed automati­ of coumarins by LC-PB/MS may offer a valid method cally by using perfluorotributylamine as the reference for evaluation of the presence of certain coumarins in compound. The repeller potential was adjusted manu­ specific plants or for monitoring variable concentra­ ally to monitor fragment ions with mass-to-charge tions when coumarins are used as pharmaceutical ratios close to sample values. The mobile phase was preparations. allowed into the ion source during calibration. The Our research group recently has modified a particle mass spectrometer was scanned from m /': 50 to 300 beam interface to make it compatible with mobile with a threshold of 50 counts. The scan speed was 1.2 phase flow rates as low as 1 ,u.L/min [29-31]. This scans per second, which gave a mean of about 10 new coupling device is fully compatible with preexist­ acquisition samples for each HPLC peak. The complete ing instrumentation and offers several advantages over mass selection report of the selected ion monitoring a conventional interface: drastic reduction in solvent (SIM) program used in this work is listed in Table 1. consumption with negligible contamination of the mass With the exception of a few compounds, to maximize spectrometer by column effluent; wider choice of sol­ the dwell time, the identification criteria were based vents and buffers that are potentially harmful to the on one characteristic ion for each substance. Peak area instrument; better signal response for high water con­ values were calculated with automatic integration. The tent mobile phases with improved sensitivity and electron energy was set at 70 eV in the positive ion chromatographic performance during gradient analy­ mode, ses; easier and more effective tunability of the interface Signal optimization with the new interface was less for chemically different mobile phases and analytes. critical and consistently easier than with the conven­ In this work an LC-PB/MS method for the analysis tional interface. A specific combination of the position of 18 different coumarins, based on a micro particle of the fused silica capillary inside the coaxial gas beam interface, is reported. A sample of Smurnium perfoliaium L. (Umbelliferae) was extracted and ana­ lyzed with this method and several coumarins were Table 1. SIM acquisition program for the analysis identified and quantified. This species can be consid­ of coumarins ered a typical example of coumarin-containing plants. Dwell time Group Channels m/z (rnsl 13 73.00 150 Experimental 208.00 150 Particle Beam Interface and Mass Spectrometer 178.00 150 202.00 150 A modified Hewlett-Packard (Palo Alto, CA) 59980B 146.00 150 particle beam interface, coupled with a Hewlett­ 148.00 150 Packard 5989A quadrupole mass spectrometer, was 162.00 150 employed. The original nebulizer was replaced by a 222.00 150 laboratory-made micronebulizer, which has been de­ 192.00 150 scribed in a previous paper [29]. The new device does 206.00 150 not require modifications to the desolvation chamber, 150.00 150 momentum separator, or their original assembly. The 120.00 150 final transfer tube prior to the ion source was in the fully retracted position. A 50-,u.m-i.d., 180-,u.m-o.d. 118.00 150 fused silica capillary tubing (Polymicro Technologies, 2 8 176.00 250 Phoenix, AZ) was used as the nebulizer tip and to 173.00 250 connect the chromatographic column. The nebulizing 246.00 250 gas was helium 5.6 purity grade (> 99.9996%) and was 186.00 250 obtained from SOL (Milano, Italy). The helium flow 160.00 250 rate was about 0.2 Lyrrun when maximum signal re­ 216.00 250 sponse was monitored by the ion source with a mobile 206.00 250 phase flow rate of 2 ,u.L/min. This value corresponds 231.00 250 to a gas pressure of 30 Ib/in.2 and to a linear velocity at the nebulizer tip of 200 my's. The gas temperature 3 2 174.00 900 was ambient while the desolvation chamber tempera- 202.00 900 134 CAPPIELLO ET AL. J Am Soc Mass Spectrom 1995,6.132-139 COMPOUND STRUCTURE COMPOUND NAME MIZ VALUES REL ABUNDANCE scoparone 206. /9/. /63 /00.50,30 0O© bydrocoumarin /48, /20, 9/ /00,80,50 OH OHQOH esculin /78, 73, /50 70, 70,50 herniarin /76, /33, /48 /00,60,50 CH: 0 OH/ oro0 -c san/omn /73, /35,246 /00,50,40 OH 0 0 CH~0!¥O fraxeun 208,59, /93 /00,50,30 OH 0 0 0 psolarene /86, 158, /02 100, 70,30 OH \o~oAo~ daphne/in /78, /50,59 /00,40,30 CH~OyO 0/900 r-methyicoumann 160, /32, 13/ /00, 70,50 OH 'GvCH, CH"Oro scopoleun /92, /77, /49 /00.60,40 I OH 0 0 0 o 00Cr0 xamhotoxin 2/6, /73,20/ 100,40,30 OH@00 umbe:lrferon /62, /34. 78 100,80,20 CH-<°ro fraxidin 222,207, /23 /00,40,20 5,7·dimethoxlcoum.