High-performance liquid chromatographic separation and quantification of the four biliverdin dimethyl ester isomers of the IX series

ANALYTICAL

BIOCHEMISTRY

101,

66-74

High-Performance Quantification

RICHARD *Department

(1980)

Liquid Chromatographic Separation and of the Four Biliverdin Dimethyl Ester Isomers of the IX Series

D. RASMUSSEN,* WALLACE H. YOKOYAMA,* SHULAG.BLUMENTHAL,*J DONALD E. BERGSTROM,? ANDBORIS H. RUEBNER* of Pathology,

School

of Medicine, Davis,

and tDepartment California 95616

of Chemistry,

University

of

California,

Received May 9, 1979 High-performance liquid chromatography (hplc) has been used to separate and quantificate the dimethyl ester (DME) derivatives of the four biliverdin isomers of the IX series: bihverdin-IXo, -IXfi, -IXy, and -1X6. Samples of 0.5 to 10.0 nmol of biliverdin DME were detected quantitatively upon elution by monitoring the absorbance at 375 nm. A technique was developed in which p-bromoacetanilide (DuPont’s recommended test compound for their Zorbax column) is used as a marker for biliverdin-IXa DME. To facilitate quantification of biliverdin-IX/3 DME, its extinction coefficient was determined. This method has been used to study biliverdin isomers in various biological species. High-resolution NMR (360 MHz) was used to further characterize the isomers.

two-dimensional analysis, or multiple development in one direction. These tic techniques provide a method of separating milligram quantities of the individual biliverdin DME isomers prepared by reductive oxidation of hemin (7). Separation schemes for biliverdin DME isomers utilizing hplc have recently been reported (12). These used reverse-phase chromatography for analytical analysis and linear gradient elution on silica for semipreparative scale separations. We describe here a rapid, sensitive, and reproducible method for the separation and quantification of the four biliverdin DME isomers of the IX series, based on isocratic elution from a silica column.

Oxidative cleavage of protoheme-IX, and loss of the iron atom, may give rise to four biliverdin isomers due to the asymmetric arrangement of substituents in protoheme-IX. These isomers have been designated biliverdin-IXa, -1X/3, -IXy, and -1X6, corresponding to opening at the (Y, p, y, or 6 methine bridge. A sensitive and reliable method for the rapid separation of these isomers is highly desirable in the study of heme catabolism by enzymatic (1,2) or nonenzymatic (3) processes. Oxidation of bilirubins to their corresponding biliverdin isomers (4-6) may make this method applicable also to the analysis of bilirubin isomers. Here possible formation of other tetrapyrroles, through dipyrrolic exchange, would have to be watched for closely (7,8). Isomer analysis based on thin-layer chromatography (tic)* (9- 11) generally requires

MATERIALS

Instrumentation. A Waters Associates (Milford, Mass.) high-pressure liquid chromatograph (Model ALC 201), equipped with a model 6000 A solvent delivery system, and a Rheodyne (Berkeley, Calif.) syringe loading sample injector (Model 7105) were used

’ To whom reprint requests should be sent. * Abbreviations used: tic, thin-layer chromatography; DME, dimethyl ester; hplc, high-performance (pressure) liquid chromatography; BV, biliverdin. 0003-2697/80/010066-09$02.00/O Copyright All rights

8 1980 by Academic Press. Inc. of reproduction in any form reserved.

AND METHODS

66

BILIVERDIN

DIMETHYL

ESTER

ISOMER

for this investigation. The column used was a 25 cm x 4.6-mm DuPont (Wilmington, Del.) Zorbax Sil-850 (5 to 6pm porous bead silica). A Varian (Palo Alto, Calif.) continuously variable uvlVis spectrophotometer (Techtron Series 635) equipped with reference and sample flow cells was used to monitor the eluate. A Linear Instruments (Irvine, Calif.; Model 252 A/MN) integrating chart recorder was used for quantitative work. Chemicals. Highly pure or repurified solvents were used for chromatography. Dichloromethane was either glass-distilled Nanograde from Mallinckrodt (Paris, Ky.) or hplc grade from Waters Associates. Methanol was glass redistilled over CaH,. The water added as a modifier was glassdistilled deionized water which was subsequently redistilled in glass over permanganate. p-Bromoacetanilide, used as a marker, was obtained in highly pure form (Aldrich, Milwaukee, Wis.; zone reflned 99.9+%). Bovine hemin was obtained from United States Biochemical Corporation (Cleveland, Ohio). Deuterated chloroform used for the NMR was 100 atom% D (Aldrich, Milwaukee, Wis.; gold label). All other chemicals were of reagent grade. Sample preparation. Biliverdin-IX DME isomers were prepared from hemin by the method of Bonnett and McDonagh (11). The four isomers, i.e., biliverdin-1X/3, -1X6, and -1Xa + IX-y, were partially purified by tic using 20 x 20-cm plates with l-mm precoated silica gel G (Analtech, Newark, Del.). Plates were developed with 5% acetone in chloroform (containing 1% ethanol). Biliverdin bands were eluted with acetone and/or methanoI, the solvent was removed under vacuum, and the residue was dissolved in chloroform. These solutions were applied on fresh silica gel G plates which were developed with heptane:2-butanone:acetic acid (10:5: 1, v/v/v). Eluted biliverdin bands were dried under vacuum. Small amounts (up to 0.5 mg) of each isomer were repurified using 0.25 mm silica gel G precoated plates (E. Merk, Darmstadt, Germany), developed

SEPARATION

AND

QUANTIFICATION

67

with the 5% acetone in chloroform solvent. Milligram quantities were crystallized from dichloromethenelpetroleum ether and the crystalline material submitted for mass and NMR spectroscopy. Separation procedures. The tic-purified biliverdin DMEs were dissolved in chloroform or methylene chloride:methanol(99: 1, v/v). The chloroform produced a spike when it passed through the detector as a nonsorbed solvent, showing the position of the solvent front. Concentrations of biliverdin solutions were approximately 1.3 X 10m3 M (0.8 &pl). For purified biliverdin DME isomer injections we used l-3 ~1. When a mixture of the four biliverdin DME isomers was injected, we used 5-10 ~1 of a mixture prepared from the purified isomers. Optimal separation was achieved with a flow rate of 1.5 mYmin, which produced a back pressure of 1100 psi. Isocratic elution with dichloromethane:methanol:water (99.0:0.9: 0.1, v/v/v) resulted in near-baseline resolution of the four isomers, within 16 min. The solvent was prepared by adding 1 part of methanol, containing 10% (by volume) water, to 99 parts of dichloromethane. VeriJcation of elution order. Verification of the elution order and of the quantitative elution of hplc-separated biliverdin DME isomers was carried out by collecting eluates after injection of the individual isomers. Eluates were collected over 1 min, from the time the isomer moved through the detector. For the F isomer, collection was for 1.5 min, to ensure total collection of this more slowly eluting isomer. Identification of the eluted compounds was done by tic. To verify that elution was quantitative, the following procedure was used. A pair of identical samples was prepared at each concentration of the four isomers. One of these was injected, chromatographed, and collected during elution (either 1 or 1.5 min). The other was diluted by collecting clean eluate into the sample for the same period of time. The chromatographed and nonchromatographed samples were then compared spectrophoto-

68

RASMUSSEN

metrically at each isomer’s Amax using the corresponding extinction coefficient (11). No published extinction coefficient for biliverdin-IXP DME was available; therefore, we determined it in the following manner. Purified biliverdin-IX@ DME was precipitated from dichloromethane/petroleum ether, the supernatant was decanted off, and the crystals were washed with petroleum ether. The sample was dried for 48 h in a vacuum desiccator over P205, three samples were weighed on an electrobalance (Cahn 21, Cahn/Ventron, Cerritos, Calif.), and the spectrophotometric absorbance was read at 382 nm (Cat-y 14, Monrovia, Calif.; Beckman Model 26, Fullerton, Calif.). The concentrations of solutions ranged from 8.5 x 10e6 to 5.4 x lo-* M. The same procedure was used to confirm the extinction coefficients reported by Bonnett and McDonagh (7,ll). Culcularions. Peak retention times were monitored by chart recorder. The following chromatographic parameters were calculated from the retention times: the capacity factor, k’; the separation factor (relative retention), a; and the resolution, Rs. The following equations were used (13): k’=-,

IR

-

10

to

a=-,

k; kl

where tR = retention time of compound and to = retention time of nonsorbed solvent. For

the

separation of two compounds factor of component 1, k; = capacity factor of component 2, and N = theoretical plate count of column. The theoretical plate count for DuPont’s Zorbax Sil-850 is greater than 9000; our column consistently measured over 12,000 using test compounds. We have used a value of N = 10,000 for our calculations. Peak areas were computed with the aid of k; = capacity

ET AL.

an integrating chart recorder, chart speed 1.O cm/min, count rate 6000 steps/min. Quantitation. Purified biliverdin DME isomer solutions were rerun on tic plates and eluted, and the eluates were dried under vacuum. The purified isomers used for quantification were diluted immediately prior to hplc separation. The solutions were kept in 5 x 25-mm glass tubes capped with latex NMR tube caps. A needle (18 gauge) was inserted through the cap. The injection syringe was inserted through the 18-gauge needle to draw samples. Another needle (25 gauge) was also inserted through the cap to let air into and out of the tube. Tubes were kept on ice and moved to room temperature close to the time of injection. Thus the samples drawn were at room temperature. All the above manipulations were done within 6 h. The concentrations of biliverdin DME isomers used were ~0.7 and 1.5 nmol/pl. A series of injections using 1, 2, 3, 4, 5, 7, and 10 ~1 for each biliverdin DME isomer were made. Intervals between injections were 1.5 min. For biliverdin-IX6 DME, injections were made at 3-min intervals. In this way no overlapping of peaks was experienced. Concentrations of solutions were calculated as described before. The ‘H NMR spectra were run on a Nicolet NT-360 FT NMR (Nicolet Technology Corporation, Mountain View, Calif.) in 100 atom% CDCl, using the CHCl, peak as a reference (6, 7.240). Chemical shifts and coupling constants for vinylic protons were determined by simulation using NTCSIM (Nicolet), a computer program derived from LAOCN III. The concentrations of each isomer were: (Y,9.8 ITIM; p, 35.5 mM; y, 7.64 mM; and 6, 19.0 mM. RESULTS

AND DISCUSSION

Figure 1 illustrates the separation of the four isomeric biliverdin DMEs of the IX series by hplc, on DuPont Zorbax Sil-850 using isocratic elution. Analysis time was

BILIVERDIN

DIMETHYL

ESTER

ISOMER

r

SF

-

I-

0

4 Time

El 12 (minutes)

16

FIG. 1. High-pressure liquid chromatogram. the four biliverdin (BV) DMEs of the IX series, isomeric mixture. Conditions: column, Zorbax Sil-850, 4.6 mm x 25 cm; mobile phase, CH,CI,:MeOH:H,O(99.0: 0.9:O. f, v/v/v); visible, 375 nm; sensitivity, 0.0-0.5 a.u.; flow, 1.5 ml/mitt; chart speed, 0.5 cmimin (Varian recorder). SF, solvent front; I, BV-IX/3 DME; 2, BV-IXa DME; 3, BV-IXy DME; 4, BV-IXG DME: 5, impurity. Injection was at time zero.

ca. 15 mm with a flow rate of 1.5 ml/min. Baseline resolution was achieved for these four isomers. The use of a more polar solvent, dichloromethane:methanol:water (98.90:0.99:0.11, v/v/v), at a flow rate of 1.5 ml/min decreased the analysis time by 3 min but led to a reduction in resolution of the biliverdin-IXcY DME and -IX/3 DME isomers. Because bilirubin-IXcr and bilirubin-IX/3 are the predominant bilirubins found in biological systems (14- 16), and because we used a quantitative method based on peak area, we felt that the optimal resolution of the corresponding biliverdin isomers justified the additional analysis time of 3 min. The preparation of biliverdin-IXa from bilirubin-IXa can lead to the production of a mixture of isomers: biliverdin-IX&, -XIIIcu, and occasionally small amounts of -111~~

SEPARATION

AND

69

QUANTIFICATION

(17). These isomers are separable as their dimethyl ester derivatives by tic (17). Biliverdin-IIIa DME was not resolved from biliverdin-IXcr with our hplc system, but biliverdin-XIIIa was well resolved from the four isomers of the IX series. Thus the purity of a biliverdin-IXa sample can be checked using the hplc technique described herein. Figure 2 illustrates the separation achieved for biliverdin-IIIa DME and -XIIIa DME when they are injected as a mixture with the four isomeric biliverdin DMEs of the IX series. Table 1 lists the retention data and calculated resolution of the separations. For high-speed separation, Rs = 1 is taken as satisfactory resolution. At Rs = 1.25 there is less than 1% band overlap for peaks of

r

I

0

I

‘I

3

6 Time

I

9 (minutes)

I

I

I2

15

FIG. 2. High-performance liquid chromatogram, the four biliverdin (BV) DMEs of the IX series and biliverdin-IIIa DME and -XIIIa DME, isomeric mixture. Conditions: column, Zorbax Sil-850 4.6 mm x 25 cm; mobile phase, CHZCI,:MeOH:H,O (99.0: 0.9:O. I, v/v/v); visible, 375 nm; sensitivity, 0.0-0.5 a.u.; flow, 1.5 mlimin; chart speed, 40 cm/h (Linear recorder). SF, solvent front; 1, BV-IXP DME; 2, BV-IXa DME coeluted with BV-IIIa DME; 3, BV-1X-y DME: 4, BV-XIIIa DME; 5. BV-IX8 DME. Injection was at ttme zero.

70

RASMUSSEN

ET AL.

TABLE

1

RETENTION TIMES (f,J, CAPACITY FACTORS (k’), SEPARATION FACTORS ((Y), AND RESOLUTION (Rs) OF THE FOUR ISOMERIC BILIVERDIN DMEs OF THE IX SERIES AND BILIVERDIN-XIIIU DME fR

Compound BV-IXP DME BV-IXa DME BV-IXy DME BV-IX8 DME

BV-XIIIa DME BV-IIIa DME

(min)

k’

7.80 8.64 10.08 14.60

2.77 3.20 3.89 6.09

1.16 1.22 1.57

2.63 3.59 7.80 (IXy vs XIIIo)

4.71 3.18

k;xr ’ k;,,,, t-1 1.21 -

8.6

a(k;lk;)

x2 of k’ values”

RS

4.69 1.65 6.68 1.58

x x x x

IO+ lO-3 lO-4 lO-3

-

3.60 -

a Five runs. DThe (Yvalue of 1.21 refers to the relative retention between biliverdin-IXy DME and biliverdin-XIIIa! DME. The Rs value of 3.60 refers to the resolution between IXy DME and XIIIa DME.

similar height (13). All isomers investigated, with the exception of biliverdin-IIIcz DME and -1Xa DME, were resolved better than 9%. Table 2 gives the mathematical relation between peak area and quantity of biliverdin DME injected using isocratic elution at 1.5 ml/min with dichloromethane:methanol: water (99.0:0.9:0.1, v/v/v) monitored at 375 nm. The mathematical relations, obtained by linear regression analysis, have slope values which correspond to their extinction coefficients in the following manner. The slope values for the 1x7, 1X/3, and IXa are very similar and decrease in the same order as their corresponding extinction coefficients; the slope value for the IXS isomer is about 15% less than the slope values of the other isomers and its extinction coefficient is also about 15% less than those of the other isomers. Our quantitative data were gathered with the detector scale set in the 0.0 to 0.5 a.u. range. In this range, high sensitivity was achieved, but noise remained less than 0.5% of full scale. Detection limits with this scale were from 0.5 nmol (peak height = 3% of full scale) to 10 nmol (peak height = 100% of full scale). Table 3 lists the absorption maxima and extinction coefficients obtained

for each of the four biliverdin isomers, as well as the values reported by Bonnett and McDonagh (11,17). Our reported extinction coefficients are an average derived from at least three weighed samples of isomer. When eluates were collected for 1 min, and the absorbance (380 nm vis) of these eluates was plotted against the quantity of biliverdin DME injected, a linear relation was obtained. Eighty percent of the injected samples was recovered, the remaining 20% was presumably lost in unTABLE MATHEMATICAL

2

RELATIONS

AND CORRELATION

COEFFICIENTS (P) OBTAINED FOR THE QUANTITATION

OF BILIVERDIN

DME ISOMERS BY hplc” Mathematical relatior#

Isomer

= = = =

9.4% + 10.8~ 11.1.x 8.56x -

0.02 0.04 0.44 0.51

No. of data points

r2

18 15 17 16

0.995 0.991 0.978 0.987

BV-IXa DME BV-IXP DME BV-IXy DME BV-IX8 DME

y y y y

a Mathematical mined from hplc mers. For details by = integrated

relations and statistical values deterquantitation of biliverdin DME isosee text and Fig. 3. area, x = nanomoles injected.

BILIVERDIN

DIMETHYL

ESTER

ISOMER

SEPARATION

TABLE SPECTROPHOTOMETRIC

ABSORPTION DETERMINED

AND

71

QUANTIFICATION

3

MAXIMA

AND

FOR BILIVERDIN

EXTINCTION

DME

COEFFICIENTS

(E,)

ISOMERS

h nmx

Isomer BV-IXo( BV-IX/3 BV-1x7 BV-IX8 u Reported

b-m CHCMI DME DME DME DME by Bonnett

380,650-656 382,644-652 376,630-638 378,

and McDonagh:

655-663

BV-IXa

swept regions of the injector. When mixed injections were used, collection of closely eluting compounds was difficult. We therefore do not recommend quantificating such samples by peak collection. A nonlinear (concave) relation was obtained for quantities in excess of 4 nmol, when peak height was plotted against quantity of biliverdin DME injected. This phenomenon, we could not explain. Chromatographic parameters vary from one hplc system to another, due to variations in system design and to changes affecting columns with time. In order to monitor these changes we have introduced the use of p-bromoacetanilide as a marker for biliverdin-IXcu DME. p-Bromoacetanilide injected alone, with biliverdin-IXcr DME, or in mixed injections with all four biliverdin IX isomers, consistently coeluted with biliverdin-IXcY. This readily available compound provides the chromatographer with a reference peak and a means of routinely checking column performance. A crude mixture of biliverdin isomers derived from the reductive oxidation of heme was obtained from the alumina column during initial sample workup and subjected to hplc analysis. Our results gave the following percentage composition on a molar basis: 26% IXp DME, 38% IXCZ DME, 15% IX6 DME, and 23% 1x7 DME. These values are in general agreement with those reported by Bonnett and McDonagh (7) and by O’Carra (18).

E"

Eli

(7), BV-IX&

52,000/15,600 52,400/15,400

51,800/15,100

52,800/16,800 45,200/15,400

54,400/18,100 43,600/15,000

and BV-IXy

(11).

We have also used this method to verify that the predominant biliverdin in chicken bile is the IXa! isomer. This was done by extracting the biliverdin from bile with chloroform, esterifying the extract with boron trifluoride/methanol, and subjecting the sample to hplc analysis. Recent advances in NMR technology have provided the means to characterize small amounts (‘1 mM) of complex molecules. As part of our program to completely characterize biliverdins and bilirubins, we have determined the ‘H NMR spectra of the four biliverdin-IX DME isomers at 360 MHz. These spectra are shown in Fig. 3. Differences between closely related structures are easily discerned in this high magnetic field. The availability of spectral data for known compounds such as these will provide a powerful tool for structure elucidation of as yet uncharacterized naturally occurring bile pigments. From the chemical shifts, reported in Table 4, the isomers are easily identified using the observations made by Bonnett and McDonagh (11). For example, it was observed that the only signals above 2.06 were singlets due to the methyl groups (Y to the lactam group. The number of singlets above 2.06 for the (Y, p, y, and p isomers are 1, 1, 0, and 2, respectively. Further, only the p and 6 isomers display two separate singlets due to the ester methyls and two separated triplets due to

a

BILIVERDIN IX Q DME

b

ElLlVERDiN IX /3DME

d

BILIVEROIN IX SDME

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

I.0 0.5 0.0 -0.5 -l.Oppm

FIG. 3. 360-MHz FT NMR of the four isomeric biliverdins of the IX series. (a) Biliverdin-IXa DME, (b) biliverdin-IX/3 DME, (c) biliverdin-1X-y DME, and (d) biliverdin-IX6 DME. For details see text and Table 4. 72

BILIVERDIN

DIMETHYL

ESTER ISOMER SEPARATION TABLE

73

AND QUANTIFICATION

4

CHEMICAL SHIFTS AND COUPLING CONSTANTS CALCULATED FOR BILIVERDIN ISOMERS FROM 360-MHz FT NMR” BV DME isomer a

P

Y

8

Aryl methyl

1.87 s, 2.07 s, 2.10 s, 2.18 s

1.78 s. 2.18 s, 2.21 s, 2.30 s

2 at 2.11 s. 2.14 s, 2.23 s

1.82 s. I.91 s. 2.15 s. 2.17 s

Ester methyl

2 at 3.66 s

3.65 s, 3.69 s

2 at 3.64 s

3.65 s. 3.69 s

Methylene

2 at 2.55 t. 2 at 2.92 t

2.51 t, 2.63 t, 2 at 2.83 t

4 at 2.55 m

2.49 t, 2.64 t, 2 at 2.82 m

mew (a,c)

6.02 s, 6.07 s

6.05 s. 6. I9 s

5.88 s, 6.00 s

5.69 s, 6.00 s

meso (b)

6.79 s

6.81 s

6.80 s

6.82 s

Vinyl 8.4 63w 8x J BCM,X

JAX J ABCMI

5.43 6.12 6.50 17.6 II.5 1.6

5.64 5.66 6.62 17.8 11.5 1.8

5.44 5.45 6.68 17.8 11.5 0.5

5.39 6.12 6.49

5.44 5.49 6.73 17.8 11.6 1.8

17.6 II.5 1.9

5.38 5.40 6.60

5.64 5.66 6.61

17.8 11.5 1.8

17.8 II.6 1.8

5.44 5.50 6.76 17.8 1I.4 1.8

” Chemical shifts in 6, residual CHCI, as standard at 7.2406, coupling constant in Hz. s = singlet, d = doublet. t = triplet, m = multiplet. 2d = superimposed doublet. Positions of vinylic protons A. B, M, and X:

R

the P-methylene groups in the two methoxycarbonylethyl side chains. In the CYand p isomers we have observed a downfield shift of the P-vinylic proton adjacent to the lactam carbonyl group from about 5.46 to 6.16. First-order methods could be used to determine the chemical shifts and coupling constants of all the vinylic protons with the exception of the chemical shifts of the P-vinylic protons of the (Y and y isomers which required computer simulation. ACKNOWLEDGMENTS The authors would like to thank Phil Burns and John Ogez of the UCD NMR Facility for their help in running spectra. This research was supported by the National Institute of Child Health, Human Development Grant IROI HD0733 1.

HB(M)

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74

RASMUSSEN

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P., and Colleran,

E. (1970) J. Chro-

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A. F. (1973) J. Chem. Sot. Perkin Trans. 1, 881-888. Schoch, S., Lempert, U., Wieschhoff, H., and Scheer, H. (1978) J. Chromatogr. 157, 357364. Snyder, L. R., and Kirkland, J. J. (1974) Modem Liquid Chromatography, Chap. 2, Wiley-Interscience, New York. Heirwegh, K. P. M., Fevery, J., Michiels, R., Van Hess, G. P., and Compernolle, F. (1975) Biochem. J. 145, 185-189. Blumenthal, S. G., Taggart, D. S., Ikeda, R. M.,

ET AL. Ruebner, B. H., and Bergstrom, D. E. (1977) Biochem.

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18. O’Carra, P. (1976) in Porphyrins and Metalloporphyrines (Smith, K. M. S., ed.), Chapter 4, ElsevierlNorth-Holland/Biomedical Press, Amsterdam. 19. Lehner, G., Braslavsky, S. E., and Schaffner, K. (1978) Liebigs Ann. Chem., 1990-2001.