Searchable libraries of MS/MS spectra, obtained using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data-dependent scan mode switching on both quadrupole ion trap and triple-quadrupole mass spectrometers in conjunction with electrospray ionization, are presented. The effects on library search scores of changing the parameters for producing collision-induced dissociation (CID) on both instrument types are systematically evaluated. These observations serve as a basis for determining a universal set of conditions for building MS/MS libraries. A group of 19 closely related steroids was used. The ability to obtain library-searchable spectra at low concentrations is demonstrated for the analysis of a sample of progesterone spiked with hydroxyprogesterone impurities at 0.1 and 0.01%.

The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.

Synthetic routes leading to the synthesis of the natural 4-phenyl, 4-propyl and 4-methyl coumarins isolated from Calophyllum sp. are presented. 4-Aryl or -alkyl, 8-and 6-acyl 5,7-dihydroxy coumarins were chromenylated and then methylated at the 5 or 7 positions. A 4-step hydrobromination-bromination4ouble dehydrobromination sequence converted the 2-met hylbutano yl side chain into the (E)-2-met hylbut -2-enoyl (tigloyl) group to give calophyllolide, oblongulide, their natural 4-propyl analogue and the corresponding regioisomers. Demethylation and cyclisation of the tigloyl group gave inophyllums C and E, tomentolides A and B, and calanolide D. Sodium boranuide reduction of the 2,3-dimethylchromanone ring afforded inophyllums A, B, D and P, soulattrolide, calanolides A-C, costatolide, and cordatolides A and B. The structures of calanolides C and D, oblongulide and apetatolide have been reassigned. The previously unknown stereochemistry about the 2,3-dimethylchromanone ring of tomentolides A and B has been established as trans. t Present address: calophyllolide 1 R = Ph 2 R=Pr 0 M e 0 Q O ' Y oblongulide 3 apetatolide 4 ponnalide 5 R = COCHMeEt calanone 6 R = COPh cordatolide A 17 from C. ~~r d a r~-o b l~n g u m ,~ are reported to all possess a 12P-hydroxy-lOP, 11 a-dimethyl substitution pattern about the chromane ring. Inophyllum P 18 isolated from C. inophyllum, l 4 calanolide B 19 from C. lanigerum, ' together with cordatolide B 21 from C. cordato-oblong~m,~ are reported to all possess the 12a-hydroxy-lop, 1 1 a-dimethyl substitution pattern. Soulattrolide 22 isolated from C. ~oulattri,'~ C.moonii,I6 and C. teysmannii," and costatolide 23 from C. costatum, ' and C. teysmannii,' are enantiomers of inophyllum P 18 and calanolide B 19, respectively. In addition, the derivatives 12-acetoxycalanolide A 15,12-methoxycalanolide A 16 and 12-methoxycalanolide B 20 have been isolated from C.lanigerum. Inophyllum A 24, isolated from C. inophyllum, ' * and C. moonii,16 and calanolide C 25, isolated from C. lanigerum," both have a 12~-hydroxy-lOf3,11 P-dimethyl substitution pattern about the chromane ring and are therefore the C-11 epimers of inophyllum B 13 and calanolide A 14, respectively. Inophyllum D 26, isolated from C. inophyllum, l o with the 12a-hydroxy-lop, 1 1 B-dimethyl substitution pattern, is View Article Online 37 R1 = Ph, R2=Me 38 R1 = Pr, R2 =Me 39 R 1 = M e , R 2 = M e 42 R1 =Ph, R2 = M e 43 R1 = Pr, R2 = M e \ / 0 HBr Br 38MeO yields of 6-and 8-acyl coumarins in the 4-phenyl series,26 and an alternative route was used for these compounds. 4-Phenyl-5,7-dihydroxy coumarin [prepared from Pechmann condensation of phloroglucinol (1,3,5-trihydroxybenzene) and ethyl benzoylacetate in 75% sulfuric was subjected to the Friedel-Crafts acylation/Fries rearrangement conditions previously described,28 to afford 8and 6-acyl coumarins 29 and 32 in 61% yield with an isomer ratio of ca. 7:4. Each pair of regioisomers was readily separated by fractional crystallisation or by chromatography as previously d e~c r i b e d .~~...

No abstract