HPLC Analysis of - American Chemical Society

8

HPLC

9

Analysis of Δ -Tetrahydrocannabinol and

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch008

Metabolites in Biological Fluids SETHR.ABBOTTandJOHNR.BERG—VarianInstrument Division, 2700 Mitchell Drive, WalnutCreek,CA94598 KAYO.LOEFFLER,SAULKANTER, and LEO E. HOLLISTER—Veterans Administration Hospital, Palo Alto, CA 94304 JOAN HAWKINS ABRAMS—California School of Professional Psychology, San Francisco, CA 94110 HUGH L. BARAS and REESE T. JONES—Langley-Porter Neuropsychiatric Institute, University of California, San Francisco, CA 94143

High performance liquid chromatography (HPLC) can rapidly separate drugs and metabolites from endogenous compounds in biological fluids. Fractions are readily collected and thus HPLC has been used in cannabinoid work to purify biological fluid extracts prior to anal ysis by techniques offering either more sensitive or specific detection than has been available for LC. HPLC cleanup of biological fluids has been reported prior to cannabinoid analysis by direct mass spectro metry, (1) GC-mass spectrometry, (Wall, this vol., Clarks, this vol.), GC (2) and radioimmunoassay (Teale, this vol.). Classical LC detectors (refractive index, fixed wavelength UV absorbance at 254 or 280 nm) have lacked the sensitivity to allow direct analysis of cannabinoids in biological fluids. However, recent develop ment of variable wavelength absorbance detectors ex tending into the 195-220nm UV region and of fluores cence detectors for HPLC led the authors to initiate 0-8412-0488-8/79/47-098-115$05.50/0 © 1979 American Chemical Society

In Cannabinoid Analysis in Physiological Fluids; Vinson, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

116

CANNABINOID ANALYSIS IN

PHYSIOLOGICAL FLUIDS

a f e a s i b i l i t y s t u d y o f d i r e c t HPLC a n a l y s i s o f c a n n a b i n o i d s i n b i o l o g i c a l f l u i d s . The s e n s i t i v i t y o f c u r r e n t HPLC d e t e c t o r s towards c a n n a b i n o i d s i s g i v e n i n T a b l e 1. T h i s paper i s a p r o g r e s s r e p o r t on t h e use o f s i m u l t a n e o u s d u a l w a v e l e n g t h (215nm, 280nm)absorbance d e t e c t i o n f o r HPLC a n a l y s i s o f c a n n a b i n o i d s i n b i o l o g i c a l f l u i d s . Work i n p r o g r e s s on f l u o r e s c e n c e d e t e c t i o n o f c a n n a b i n o i d s w i l l be t h e s u b j e c t o f a l a t e r r e p o r t . TABLE


HPLC

Detector

Detection

of

Cannabinoids

Sensitivity* in abscence o f m a t r i x interference

R e f r a c t i v e Index

300

254,280 nm

10 ng (THC 1 ng (CBN

215

0.5

nm

I

ng type) type)

ng

Fluorescence A. N a t i v e Fluorescence

1 ng p r e d i c t e d

B. D a n s y l derivatives

5 pg (3)

Selectivity vs.Endogenous Substances None Discriminates against l i p i d s , which do n o t absorb a t 280 nm ^300-fold d i s crimination against l i p i d s

Excellent

Interference from b i o l o g i c a l phenols, ίο and 2° amines. Amines interference can be s e r i o u s * S e n s i t i v i t i e s based on 2 0 0 y l peak volumes o b t a i n e d u s i n g 25 cm χ 2.1 mm ID a n a l y t i c a l HPLC columns.


8.

ABBOTT E T A L .

117

HPLC Analysis

EXPERIMENTAL


Recovery

o f Cannabinoids

from

Human

Urine

A v o l u m e o f u r i n e c o n t a i n i n g 25mg o f c r e a t i n i n e was h y d r o l y z e d w i t h b e t a - g l u c u r o n i d a s e / a r y l s u l f a t a s e and c o n c e n t r a t e d i n a r o t a r y e v a p o r a t o r ( 5 ) . The c o n c e n t r a t e , d i l u t e d t o 1 0 m l w i t h d i s t i l l e d w a t e r , was a d j u s t e d t o pH 8, t h e n e x t r a c t e d w i t h h e x a n e , a d j u s t e d t o pH 12 a n d e x t r a c t e d w i t h e t h e r , a n d t h e n a d j u s t e d t o pH 2.5 a n d e x t r a c t e d w i t h e t h e r a g a i n . The l a t t e r e t h e r e x t r a c t was e x t r a c t e d t h r e e t i m e s w i t h 8 m l o f 2% sodium b i c a r b o n a t e (pH 8 . 8 ) . T h e e t h e r p h a s e , c o n t a i n i n g t h e m o r e p o l a r a c i d i c m e t a b o l i t e s , was a d j u s t e d t o pH 2.5 a n d e x t r a c t e d w i t h e t h e r ( 5 ) . Each o f the o r g a n i c e x t r a c t s was e v a p o r a t e d u n d e r n i t r o g e n . The r e s i d u e s w e r e t h e n r e c o n s t i t u t e d i n 50 y l m e t h a n o l . A s c h e m a t i c d e s c r i p t i o n o f t h e e x t r a c t i o n scheme i s g i v e n i n F i g u r e 1. Studies with urines spiked with radiol a b e l l e d s t a n d a r d s h a v e shown t h e c a n n a b i n o i d s t o p a r t i t i o n as f o l l o w s : h e x a n e e x t r a c t = THC, CBN, CBD, m o n o h y d r o x y metabolites E-I e x t r a c t = Polyhydroxy metabolites E-II extract = Weakly p o l a r a c i d s (e.g. A -THC11-oic acid) E-III extract = More p o l a r a c i d s 9

Recovery

of Cannabinoids

f r o m Human B r e a s t

Milk

A 4 m l s a m p l e o f m i l k was h y d r o l y z e d a n d e x t r a c t e d according t o the urine procedure. The hexane e x t r a c t r e s i d u e was d i s s o l v e d i n 2 m l m e t h a n o l a n d c e n t r i f u g e d at ambient temperature, removing white l i p i d m a t e r i a l . T h e m e t h a n o l was e v a p o r a t e d u n d e r n i t r o g e n a n d t h e r e s i d u e r e c o n s t i t u t e d i n 50 y 1 h e x a n e .

HPLC L i q u i d C h r o m a t o g r a p h y was p e r f o r m e d o n a V a r i a n 8520 d u a l s y r i n g e pump l i q u i d c h r o m a t o g r a p h . Two V a r i c h r o m ™ v a r i a b l e w a v e l e n g t h d e t e c t o r s were u s e d i n s e r i e s t o provide a dual wavelength d e t e c t i o n system. A 25cm χ 2.1 mm ID V a r i a n M i c r o P a k MCH-10 c o l u m n , p r e p a r e d by c h e m i c a l l y bonding a monomolecular l a y e r o f


118

CANNABINOID

ANALYSIS

I N PHYSIOLOGICAL

FLUIDS

ENZYME-HYDROLYZED URINE (pH 5.5)

I

Extract with hexane at pH 8


Adjust to pH 12 Extract with ether

Adjust to pH 2.5 extract with ether Aqueous (discard)

Ether soluble (E-l)

Ether-soluble wash with2% NaHC0 (pH8.8) 3

Adjust to pH 2.5) extract with ether Aqueous (discard)

Hexane-soluble ~~~~~~~

Ether-soluble (Ε-Ill)

Ether-soluble (E-ll)

Hexane E-l E-ll Ε-Ill

THC, CBN, CBD, monohydroxys polyhydroxys polar acids (ll-oic) highly polar acids

Figure 1. Schematic of extraction scheme for cannabinoids in biological fluids

o c t a d e c y l o n t o 10 m i c r o n s i l i c a g e l , was used f o r t h e s e p a r a t i o n o f the u r i n e e x t r a c t s . A s i m i l a r s i z e V a r i a n M i c r o P a k CN-10 column, p r e p a r e d by c h e m i c a l l y b o n d i n g a monomolecular l a y e r o f c y a n o p r o p y l groups o n t o 10 m i c r o n s i l i c a g e l was u s e d f o r s e p a r a t i o n o f t h e m i l k extract. ANALYSIS OF URINE Results U r i n e samples c o l l e c t e d from two human s u b j e c t s , p r i o r t o (minus 2 4 t o 0 hours) and a f t e r ( p l u s 2 t o 6 hours) o r a l a d m i n i s t r a t i o n o f 30 mg A - T H C , were h y d r o l y z e d and e x t r a c t e d as d e s c r i b e d i n t h e e x p e r i m e n t a l s e c t i o n . P r e - and p o s t - d r u g e x t r a c t s c o r r e s p o n d i n g t o e q u i v a l e n t u r i n a r y c r e a t i n i n e l e v e l s were s e p a r a t e d by r e v e r s e phase HPLC. The p r e - d r u g e x t r a c t was used as a 9


ABBOTT E T A L .


8.

HPLC Analysis

119

"blank", c h a r a c t e r i s t i c o f the endogenous c o m p o s i t i o n of the s u b j e c t ' s u r i n e . A M i c r o P a k ™ MCH-10 r e v e r s e phase column was chosen f o r s e p a r a t i o n o f the hexane and e t h e r e x t r a c t s . The monomolecular C^g bonded phase p r o v i d e s e f f i c i e n t s e p a r a t i o n o f both p o l a r and n o n - p o l a r s u b s t a n c e s and r a p i d e q u i l i b r a t i o n to i n i t i a l a c t i v i t y a f t e r g r a d i e n t e l u t i o n programs. The r e v e r s e phase column p r o v i d e s s y m m e t r i c a l , narrow peaks f o r the c a n n a b i n o i c a c i d s , which t e n d t o t a i l on p o l a r , normal phase columns (e.g. silica). Use o f a w a t e r - a c e t o n i t r i l e mobile phase system a l l o w e d d e t e c t i o n below 220nm; the A9-THC e x t i n c t i o n c o e f f i c i e n t i s c a . 30 f o l d g r e a t e r a t 215nm than 280nm (and the m a t r i x i n t e r f e r e n c e was l e s s a t 215nm). Thus, A -THC d e t e c t i v i t y o f the HPLC system w i t h 215nm d e t e c t i o n was s u p e r i o r t o t h a t a t 2 80nm by an o r d e r o f mag nitude . Endogenous substances i n the e x t r a c t s a r e more p o l a r than the c a n n a b i n o i d s and e l u t e b e f o r e them on the r e v e r s e phase column. On p o l a r , normal phase columns, s t r o n g a d s o r p t i o n o f endogenous s p e c i e s r e q u i r e s p e r i o d i c column c l e a n - u p . T h i s p r o b l e m was n o t e n c o u n t e r e d w i t h the r e v e r s e phase g r a d i e n t system. S e p a r a t i o n s were m o n i t o r e d w i t h v a r i a b l e wave l e n g t h d e t e c t o r s s e t a t 215nm and 2 80nm r e s p e c t i v e l y . The A215/A2Q0 absorbance r a t i o a c t s as a v a l u a b l e check on e x t r a c t peaks h a v i n g r e t e n t i o n times c o i n c i d e n t w i t h cannabinoid standards. The A 2 1 5 / A 2 8 O r a t i o s o f s e v e r a l c a n n a b i n o i d s t a n d a r d s l a r e l i s t e d i n T a b l e 2. The can n a b i n o i d s s e g r e g a t e i n t o two c l a s s e s b a s e d on the A 2 1 5 / A28O r a t i o . The THC c l a s s has a h i g h absorbance r a t i o (24-37), r e f l e c t i n g the r e l a t i v e l y weak 280nm absorb ance ( ε - 1500) o f the p h e n o l i c r i n g . The CBN c l a s s has a low absorbance r a t i o (- 2) r e f l e c t i n g the s t r o n g 280nm absorbance ( ε -18,000) o f a hydroxybipheny1 r i n g system. 9

1.

Hexane

Extract*

Subject

1

The 215nm chromatograms o f the p r e - and p o s t - d r u g hexane e x t r a c t s e q u i v a l e n t t o 5 mg c r e a t i n i n e (29 ml u r i n e ) a r e shown i n F i g u r e 2. In t h e 5%B/min .water •+ a c e t o n i t r i l e g r a d i e n t , a v a i l a b l e s t a n d a r d s r a n g i n g from the r e l a t i v e l y p o l a r A - T H C - l l - o i c a c i d t o the n o n p o l a r A -THC e l u t e d between -10-14 minutes (50+70% a c e t o n i t r i l e ) . More p o l a r c a n n a b i n o i d s such as hydroxy a c i d s 9

9

These r a t i o s a r e s e n s i t i v e t o d e t e c t o r c a l i b r a t i o n and s h o u l d always be determined on a g i v e n d u a l d e t e c t o r system by m o n i t o r i n g the chromatography o f s t a n d a r d s .


CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

CD


2-6 Hours Post-Drug

-24 to Zero Hours (Pre-Drug)

I

1

1

0

4

8

1

12

1

16

1

20

1

Γ

24 28

TIME (MINUTES) Figure 2. The 215-nm chromatograms of hexane extracts of urine of subject I , pre- and post-dose. A MicroPak MCH-10 column with 1 mL/min linear gradient program from water -» acetonitrile at + 5 % B/min.


8. ABBOTT E T A L .

HPLC Analysis

121

TABLE ^•215^280

Absorbance

Cannabinoid

Ratios

of

Cannabinoids

Absorbance R a t i o

9

Δ -THC A -THC CBD 8B-OH-A -THC 11-0H-A -THC 8, 11-di-OH -A -THC A - l l - o i c Acid CBN CBN-ll-oic Acid

28.6 23.8 35.9 36.5 37.0 35.2 28.1 2.2 1.7

9

9

9

9

9


2

THC C l a s s

CBN

Class

should e l u t e e a r l i e r , The 5-14 minute (25+70% a c e t o n i t r i l e ) r e g i o n was thus e s t i m a t e d t o be t h e c a n n a b i n o i d e l u t i o n r e g i o n . T h i s r e g i o n was s c r e e n e d f o r peaks w i t h r e t e n t i o n t i m e s and absorbance r a t i o s c o i n c i d e n t w i t h a v a i l a b l e s t a n d a r d s and f o r peaks n o t m a t c h i n g r e t e n t i o n t i m e s o f s t a n d a r d s b u t h a v i n g THC o r CBNtype r a t i o s . A peak was o b s e r v e d c o i n c i d e n t w i t h t h e A -THC r e t e n t i o n time i n t h e p o s t - d r u g e x t r a c t chromatogram. The THC r e g i o n was b l a n k i n t h e p r e - d r u g e x t r a c t chromatogram. The 215 and 2 80nm chromatograms o f t h e p o s t - d r u g e x t r a c t a r e shown i n F i g u r e 3. The s u s p e c t e d A -THC peak had an â ^ ^ / ^ g Q r a t i o o f 2 8-33. The u n c e r t a i n t y i n measurement was due t o t h e weakness o f t h e 2 80nm peak. The A -THC s t a n d a r d had a r a t i o o f 2 8.6. The peak was thus a s s i g n e d t o A9-THC. The peak absorbance and 215nm e x t i n c t i o n c o e f f i c i e n t o f A9-THC i n d i c a t e 51ng i n j e c t e d o n t o t h e column, w h i c h e x t r a p o l a t e s t o 1.26 yg and 1.7 ppb A -THC i n t h e +2 t o 6 h o u r u r i n e (125mg c r e a t i n i n e , 719ml u r i n e ) . A peak was o b s e r v e d i n t h e c a n n a b i n o i d r e g i o n o f the p o s t - d r u g e x t r a c t w h i c h had a CBN-type r a t i o o f 2. The r e g i o n was c l e a r i n t h e p r e - d r u g e x t r a c t . The peak r e t e n t i o n time d i d n o t match any o f t h e a v a i l a b l e s t a n d a r d s . I t s e l u t i o n time s u g g e s t s t h a t i t i s l e s s p o l a r than 83-OHA -THC and s l i g h t l y more p o l a r t h a n CBD. 9

9

9

9

9

1

The r e t e n t i o n o r d e r o f t h e n a t u r a l , n e u t r a l c a n n a b i n o i d s on t h e r e v e r s e d phase column i s CBD-CBN-THC w i t h the THC t h e most r e t a i n e d .


122



2-6 Hours Post -Drug

R

0

1

1

4

8

'

1

1

1

1

12

16

20

24

- Γ -

28

TIME (MINUTES)

Figure 3. The 215- and 280-nm chromât ο grams of post drug hexane extract of urine of subject 1. Micro?ak MCH-10 column with 1 mL/min linear gradient program from water —> acetonitrile at +5% B/min.

2.

Hexane

Extract,

Subject

2

A hexane e x t r a c t o f t h e u r i n e o f a second s u b j e c t was a n a l y z e d t o see i f t h e C B N - c l a s s peak r e p e a t e d . The p r e - and p o s t - d r u g hexane e q u i v a l e n t s t o 5mg c r e a t i n i n e o f a n o t h e r s u b j e c t were s e p a r a t e d u s i n g a s l o w e r g r a d i e n t (3%B/min) t h a n t h a t used f o r S u b j e c t 1. The 215nm c a n n a b i n o i d e l u t i o n r e g i o n i s shown i n F i g u r e 4B, s i d e by s i d e w i t h t h a t o f t h e S u b j e c t 1 u r i n e i n F i g u r e 4A. The 3%B/min g r a d i e n t gave somewhat b e t t e r r e s o l u t i o n o f endogenous m a t e r i a l from t h e c a n n a b i n o i d r e g i o n and i s t h e p r e f e r r e d program f o r t h e hexane e x t r a c t .


8.

ABBOTT E T A L .

123

HPLC Analysis

SUBJECT 2 -24 TO ZERO HRS. P R E - D R U G URINE 5 mg CREATININE OUT OF 462

SUBJECT 1


-24 TO ZERO HRS. P R E - D R U G URINE 5 mg CREATININE OUT OF 124

+2-6 H O U R S POST-DRUG

+2-6 HOURS POST-DRUG

Figure 4. The 215-nm chromât ο grams of hexane extracts of urines of subjects 1 and 2, pre- and post-dose. Micro?ok MCH-10 column with 1 mL/min linear gradient program from water —» acetonitrile at 3% B/min.

A g a i n , a peak was o b s e r v e d i n t h e S u b j e c t 2 p o s t d r u g u r i n e c o i n c i d e n t w i t h t h e A -THC r e t e n t i o n t i m e . I t s absorbance r a t i o was g r e a t e r t h a n 20. A c l o s e r e s t i m a t e was p r e c l u d e d by t h e weakness o f t h e 280nm ab sorbance. I t s e l u t i o n r e g i o n was b l a n k i n t h e p r e - d r u g u r i n e . The peak was t h u s a s s i g n e d t o A -THC and c o r responds t o 15ng i n j e c t e d o n t o t h e column, w h i c h e x t r a p o l a t e s t o 1.39 yg and 4 ppb A -THC i n t h e +2-6 hour u r i n e (462mg c r e a t i n i n e , 347ml u r i n e ) . 9

9

9


124



y

The HPLC e s t i m a t e s o f t h e A -THC l e v e l s , based on t h e 215nm e x t i n c t i o n c o e f f i c i e n t , c o r r e s p o n d t o e x c r e t i o n o f 0.004 and 0.005% o f dose i n t h e +2 t o 6 hour u r i n e s o f s u b j e c t s 1 and 2. These l e v e l s agree w i t h p r e v i o u s TLC a n a l y s e s o f o t h e r u r i n e s o f t h e s e p a t i e n t s . As was t h e case i n t h e p o s t - d r u g e x t r a c t o f sub j e c t 1, a peak w i t h a C B N - c l a s s absorbance r a t i o o f 2 was o b s e r v e d i n t h e c a n n a b i n o i d e l u t i o n r e g i o n o f sub j e c t 2. I t s e l u t i o n r e g i o n was b l a n k i n t h e p r e - d r u g u r i n e . The r e t e n t i o n t i m e a g a i n s u g g e s t s a compound s l i g h t l y more p o l a r t h a n CBD. The appearance o f t h i s C B N - c l a s s peak i n t h e p o s t d r u g u r i n e s o f b o t h s u b j e c t s and c o n c o m i t a n t absence i n t h e p r e - d r u g u r i n e s s u g g e s t s a CBN-type m e t a b o l i t e o f Δ9 -THC. The m e t a b o l i t e r e t e n t i o n t i m e s u g g e s t s a r e l a t i v e l y n o n p o l a r s i d e - c h a i n h y d r o x y l a t e d CBN (I) o r a c a n n a b i n o d i o l type s t r u c t u r e (II) .

(i)

(II).


8.

HPLC Analysis

125

The c a n n a b i n o d i o l h y p o t h e s i s i s i n t e r e s t i n g i n t h a t p r e v i o u s gas chromatography-mass fragmentography (GC-MF) a n a l y s e s o f s i m i l a r u r i n e s from our l a b (9) c o n s i s t e n t l y d e t e c t e d a compound h a v i n g a GLC r e t e n t i o n time c a . 8% g r e a t e r t h a n t h a t o f CBN, b u t w i t h t h e c h a r a c t e r i s t i c mass f r a g m e n t s o f CBN. C a n n a b i n o d i o l was ob s e r v e d by van Ginneken e t a l ( 1 0 M 7 ) t o have a c a n n a b i n o l l i k e mass f r a g m e n t a t i o n p a t t e r n . I t s h o u l d be n o t e d t h a t van Ginneken d i d n o t i s o l a t e c a n n i b i n o d i o l f o r s p e c t r a l a n a l y s i s and t h u s one i s n o t c e r t a i n o f i t s 215/ 280 r a t i o . I t i s p o s s i b l e t h a t s t e r i c hindrance o f the i s o p r o p e n y l and n e a r b y p h e n o l i c h y d r o x y l c o u l d reduce c o - p l a n a r i t y o f the b i p h e n y l r i n g s , a l t e r i n g the absorbance r a t i o from t h a t e x p e c t e d f o r a CBN compound. I f t h e u n i d e n t i f i e d peak had a c h a r a c t e r i s t i c CBN e x t i n c t i o n c o e f f i c i e n t a t 215nm ( ε ^ 3 7 , 5 0 0 ) , one c a l c u l a t e s i t s l e v e l t o be 0.7ppb i n t h e u r i n e o f s u b j e c t 1 and 21ppb i n t h e u r i n e o f s u b j e c t 2.

A


ABBOTT E T A L .

3.

A

EII

Extract

3

Subject

1

The 215nm chromatograms o f t h e p r e - and p o s t - d r u g E I I e t h e r e x t r a c t s (see F i g u r e 1) e q u i v a l e n t t o 2.5mg c r e a t i n i n e a r e shown i n F i g u r e 5. The E I I e x t r a c t i s o f i n t e r e s t because o f r e c e n t work by Greene ( t h i s v o l ume) i n w h i c h t h e r a t i o o f bound t o unbound u r i n a r y A 9 - T H C - l l - o i c a c i d , t h e major known THC m e t a b o l i t e i n humans, was p r o p o s e d t o be a f u n c t i o n o f time a f t e r d o s e . As d e s c r i b e d i n t h e e x p e r i m e n t a l s e c t i o n , t h i s a c i d p a r t i t i o n s i n t o the E I I f r a c t i o n . A peak was o b s e r v e d c o i n c i d e n t w i t h t h e r e t e n t i o n time o f Δ9-THC-ll-oic a c i d , h a v i n g an a b s o r b a n c e r a t i o o f 26-32 i n t h e p o s t - d r u g e x t r a c t . I t s e l u t i o n a r e a was b l a n k i n t h e p r e - d r u g chromatogram. The peak was t h u s a s s i g n e d t o A - T H C - l l - o i c a c i d and r e p r e s e n t s 98ng i n j e c t e d , w h i c h e x t r a p o l a t e s t o 4.85 yg and 6.7ppb i n t h e +2-6 hour u r i n e . S i l i c a g e l TLC a n a l y s i s o f t h i s u r i n e e x t r a c t i n d i c a t e d a Δ -11o i c a c i d l e v e l o f c a . 6 yg. The TLC l e v e l was ob t a i n e d by v i s u a l e s t i m a t i o n o f t h e F a s t B l u e S a l t (B) (FBSB) s p o t i n t e n s i t y . The agreement o f HPLC and TLC was t h u s e x c e l l e n t . The r e l a t i v e l y l a r g e e x c r e t i o n o f A - T H C - l l - o i c a c i d i n humans and t h e low 215nm m a t r i x l e v e l o f t h e E I I e t h e r e x t r a c t makes r a p i d , d i r e c t HPLC-215nm ab s o r b a n c e a n a l y s i s r e l a t i v e l y s i m p l e . The f a s t 5%B/min g r a d i e n t program (20 m i n u t e a n a l y s i s ) i s a c c e p t a b l e for t h i s application. 9

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HPLC Analysis of - American Chemical Society

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