Quantitative Determination of the Enantiomeric Composition of Panthotenic Acid Solutions : A Mass Spectrometry Experiment

Rapid and quantitative determinations of the enantiomeric compositions of pantothenic acid solutions were carried out by using electrospray ionization tandem mass spectrometry and applying the kinetic method to perform the data analysis. Deviations of ca. 1% between the actual and experimental enantiomeric compositions were observed.


Introduction
Pantothenic acid belongs to the group of B vitamins and, as a constituent of coenzyme A, plays a key role in the metabolism of carbohydrates, proteins and fats, and is therefore important for the maintenance and repair of all cells and tissue.It is involved in reactions that supply energy, in the synthesis of vital compounds as sterols (cholesterol), hormones (growth, stress and sex hormones), neurotransmitters (acetylcholine), phospholipids (components of cell membranes), porphyrin (component of hemoglobin, the oxygen-carrying red blood cell pigment), antibodies, and in the metabolism of drugs (sulfonamides).It also stimulates the adrenal glands and increases the production of cortisone for healthy nerves and skin.It has been verified, however, that only the Denantiomer, its naturally-occurring form, has vitamin activity.The L-form, L-pantothenic acid, has been shown to have an antagonistic effect in animal studies. 1 There are few issues in chemistry that has drawn as much attention as the chiral nature of molecules.For instance, systematic examination of the biological activity of individual enantiomers is the rule for all new racemic drug candidates, and an increasing number of optically pure drugs has been approved and marketed. 2oteworthy advance has been made during the past few years on general methods of chiral identification and quantification 3 based entirely on mass spectrometry.It is possible to group the methods used for chiral recognition and for quantification of enantiomeric excess by mass spectrometry into four wide types: (a) generation of hostguest diasteromeric adducts using a chiral guest; 4,5 (b) determination of rates of ion/molecule reactions between an enantiomeric guest and a host molecule; 6 (c) collisioninduced dissociation (CID) of diasteromeric adducts in a MS/MS experiment; 7 (d) utilization of the kinetic method [8][9][10][11][12] to quantify the MS/MS chiral effect. 13This last methodology has been successfully applied to quantitative analysis of aminoacids, 13,14 α-hydroxyacids, 15 sugars 16 and some drugs [17][18][19] and has been shown to be fast and to require very small amounts of sample.
The chiral distinction based on the kinetic method approach, the subject of this paper, involves, as the first step, the choice of systems that can promote acceptable enantiomeric distinction.In order to do that, the singly-charged -formed in an electrosprayed solution containing a chiral analyte (A D or A L , respectively), a chiral reference compound (ref * ), and a metal cation (usually M 2+ ) -are mass-selected and fragmented upon collision-induced dissociation.Scheme 1 displays the competitive dissociation of both trimeric complexes to form the corresponding dimeric fragments, i.A L ) -H] + results in differences in their rates of formation and hence in the relative abundance ratios (R D or R L ) for the D and L isomers, defined in equations 1 and 2, respectively: The ratio of the individual ratios, R D to R L , defined as R chiral (equation 3), indicates the level of chiral distinction achievable in a particular experiment.
Evidently, when R chiral = 1 there is no chiral discrimination, which denotes that this specific array of metal and chiral reference is not able to originate effective enantiomeric distinction under these specific conditions.Therefore, the best systems to be selected are those ones that provide R chiral as far as possible from unity, provided that accurate abundance ratios can still be measured.It must be mentioned, however, that the selection of adequate systems for enantiomeric distinction has been exclusively performed, as recently reported in literature, 13,16,19 based on an alternative procedure, i.Thus, unknown enantiomeric mixtures are then analysed by measuring the ratio of the two fragment ions, i.e. [M(ref*)(A) -H] + ] and M(A) 2 -H] + ), in a single tandem mass spectrum.This methodology is already wellestablished and known as the single ratio method. 12he aim of this paper is, therefore, to apply the kinetic method strategy to determine the enantiomeric composition of pantothenic acid solutions, an important substance for the pharmaceutical and food industries.As chiral reference compounds, a series of α-hydroxyacids (L-tartaric acid, L-lactic acid, L-3-phenyl lactic acid, L-citramalic acid, L-mandelic acid, L-malonic acid, L-citric acid), sugars (D-glucose, D-mannose, D-galactose), aminoacids (L-Pro, L-Trp, L-Ala, L-Arg, L-Val, L-Tyr, L-Glu, L-Phe, L-Cys, L-Leu, L-His, L-Lys), and modified aminoacids (N-t-Boc-L-Pro, N-t-Boc-L-Phe or N-Fmoc-L-Pro) were tested in order to select the best system for chiral recognition and quantitation.These references were chosen since (a) they can be easily obtained in enantiomerically pure form; and (b) in previous studies, some of these compounds proved to be useful as reference compounds in chiral analysis. 15,17,18 F17][18][19]

Experimental
All experiments were performed using a commercial LCQ Advantage ion trap mass spectrometer (Thermo Finnigan, San Jose, CA), equipped with an ESI source and operated in the positive ion mode.The mass spectra reported are the average of about 40 scans, each requiring 0.2 s.Samples were infused into the ESI source via a syringe pump at a flow rate of 2.00 µL min -1 .Typical ESI conditions were as follow: heated capillary temperature, 150 o C; sheath gas (N 2 ) flow rate, 0.75 L min -1 ; spray voltage 5 kV; capillary voltage 3 V; tube lens off set voltage, 40 V.The aqueous methanol 1:1 solutions were prepared by the mixture of the following reagents: (a) 2x10 -5 mol L -1 of calcium pantothenate, the calcium salt of pantothenic acid (in these experiments it was used the pure D-enantiomer and the racemic mixture of such reagent); (b) 2x10 -5 mol L -1 of a chiral reference compound (ref*) such as: α-hydroxyacids (L-tartaric acid, L-lactic acid, L-3-phenyl lactic acid, L-citramalic acid, L-mandelic acid, L-malonic acid, L-citric acid), sugars (D-glucose, D-mannose, D-galactose), aminoacids (L-Pro, L-Trp, L-Ala, L-Arg, L-Val, L-Tyr, L-Glu, L-Phe, L-Cys, L-Leu, L-His, L-Lys), and modified aminoacids (N-t-Boc-L-Pro, N-t-Boc-L-Phe or N-Fmoc-L-Pro); and (c) 2x10 -5 mol L -1 of a metal cation (nitrate salt of Ca 2+ , Cu 2+ , Ni 2+ , Zn 2+ ). Figure 2 (a and b) shows the collision-induced dissociation spectrum of the mass-selected trimeric complexes [Ca(N-Fmoc-L-Pro)(D-pantacid) 2 -H] + (formed in the solution containing pure D-pantothenic acid) and [Ca(N-Fmoc-L-Pro)(DL-pantacid) 2 -H] + (from a solution with racemate pantothenic acid), respectively.The R chiral for this system, calculated by using equation 3, was found to be 0.71 and reflects the difference in stability of the diastereomeric ions

Results and Discussion
), which determination is necessary to calculate R chiral , is related to the dissociation of the trimeric complex [Ca(N-Fmoc-L-Pro)(L-pantacid) 2 -H] + (hypothetically formed in a solution containing pure L-pantothenic acid) and is obtained by extrapolation to zero in a plot of ln(R) versus the molar fraction of Dpantothenic acid, as shown in Figure 3.
Curiously, the trimeric complex [Ca(N-Fmoc-L-Pro) 2 (pantacid) -H] + (m/z 932), which could alternatively be used for such chiral distinction (see some considerations about it in Introduction section), did not produce a convenient value for R chiral .Moreover, the use of other systems, i.e. different combinations of the reference compounds and metal cations cited in the Experimental section, did not furnish satisfactory results for the chiral recognition of pantothenic acid solutions.This was caused by at least one among the following reasons: A linear relationship between ln (R), R = [Ca(N-Fmoc-L-Pro)(pantacid) -H] + / [Ca(pantacid) 2 -H] + ), versus the molar fraction of D-pantothenic acid is observed with an excellent correlation coefficient (R 2 = 0.9995) (Figure 3).The ratio R for any solution containing Ca 2+ , N-Fmoc-L-Pro, and panthotenic acid can be easily determined by measuring the intensities of the dimeric complexes [Ca(N-Fmoc-L-Pro)(pantacid) -H] + (m/z 595) and [Ca(pantacid) 2 -H] + (m/z 477) formed from the dissociation of the mass-selected trimeric complex [Ca(N-Fmoc-L-Pro)(pantacid) 2 -H] + (m/z 814), as exemplified in Figures 2a and 2b.Using the calibration curve in Figure 3, the enantiomeric composition of prepared solutions of pantothenic acid was determined and outstanding results were obtained, as illustrated by the data of Table 1.Significantly, it was verified that the relative   concentrations of pantothenic acid vs. N-Fmoc-L-Pro did not affect the chiral discrimination in these systems. 16,20 is is an indispensable result for successful quantitative analysis of unknown samples.

Conclusions
The subject reported in this paper is of wide relevance, particularly to the pharmaceutical and food industries, both for the general demonstration of a simple method of enantiomer quantitation and in particular because pantothenic acid represents a valuable substance with improved possibilities of application.Furthermore, the present study demonstrates for the first time that Ca 2+ , a non-transition metal previously tested in similar chiral recognition experiments, 21 has a better performance than transition metal cations, such as Cu 2+ , Ni 2+ , Zn 2+ .The results presented herein demonstrate that not only [M(ref*) 2 (A) -H] + but also [M(ref*)(A) 2 -H] + trimeric complexes can be used for chiral quantitation by the single ratio method.It can also be envisioned that once the linearity of the semilog plot has been established for a chiral analysis, a twopoint calibration curve would allow a faster but still reliable method of quantitative chiral analysis.By the use of a two-point calibration curve, which could be built by using the D-enantiomer and the racemic mixture of pantothenic acid, the enantiomeric composition of unknown solutions could be determined in a quick, but still reliable, way.
Scheme 2 shows the energy diagram for the competitive dissociations of the trimeric complexes [M(ref*)(A D ) 2 -H] + and [M(ref*)(A L ) 2 -H] + to form the corresponding dimeric fragments.Note that the dimeric complexes [M(A D ) 2 -H] + and [M(A L ) 2 -H] + are enantiomers and, therefore, possess exactly the same energy level.

Figure 1 Figure 1 -
Figure 1 shows the ESI-MS spectrum of a solution containing calcium D-pantothenate and N-Fmoc-L-Pro.The formation of the trimeric complex [Ca(N-Fmoc-L-Pro)(D-pantacid) 2 -H] + (m/z 814) is observed and indicated in this spectrum.Note that several other ionic adducts, such as [D-pantacid + H] + (m/z 220), [Ca(NFmoc-L- (a) no formation of the required trimeric complexes [M(ref*)(A) 2 -H] + or [M(ref*) 2 (A) -H] + ; (b) no production of both dimeric ions during the dissociation of such trimeric complexes; and (c) systems with R chiral very close to unit.

Figure 3 -
Figure 3 -Calibration curve for chiral analysis of pantothenic acid using Ca 2+ as the metal cation and N-Fmoc-L-Pro as the chiral reference compound.The chiral selectivity factor (R chiral ) is 0.71 and the correlation coefficient (R 2 ) is 0.9995.Each point represents an average of ten measurements.

Table 1 .a
Actual and experimental values for the enantiomeric composition of pantothenic acid solutionsFraction of D-pantothenic acid (Values obtained from the calibration curve (Figure3); b Average of ten measurements (95% confidence interval).