NBSIR 79-1947

Development of a Standard

eference Material for

ngiotensin I

Canter for Anatytical Chemistry

National Measurement Laboratory

National Bureau of Standards

Washington, D.C. 20234

-3

December 1979

Report for Period

March 1 977 to March 1 979

Final Report on Agreement #Y01-HV- 70042

Prepared for National Heart, Lung and Blood Institute

National Institutes of Health

Bethesda, Maryland 20014

NBSIR 79-1947

DEVELOPMENT OF A STANDARD

REFERENCE MATERIAL FOR

ANGIOTENSIN I

S. A. Margolis and R. Schaffer

Center for Analytical Chemistry

National Measurement Laboratory

National Bureau of Standards

Washington, D.C. 20234

December 1979

Report for Period

March 1 977 to March 1 979

Final Report on Agreement #Y01-HV-70042

Prepared for National Heart, Lung and Blood Institute

National Institute of Health

Bethesda, Maryland 20014

U.S. DEPARTMENT OF COMMERCE.

Luther H. Hodges, Jr., Under Secretary

Jordan J. Baruch, Assistant Secretary for Science and Technology

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Report on Agreement irYOl -HV- 70042

for the period

March 1977 to March 1979

Issued December 1979

S. A. Margolis and, R, Schaffer

Prepared for

National Heart, Lung and Blood Institute

National Institutes of Health

Bethesda, Maryland 20014
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Final Report on Agreement rfYOl-HV- 70042

Development of a Standard Reference Material

for Angiotensin I

S. A. Margolis and R. Schaffer

Table o£ Contents

Page

Introduction 1

Methods for Analysis of Peptide Purity

A. Resolution of Peptides with Amino Acid Composition

Similar to Angiotensin I 3

B. Resolution of Truncated Peptides Related to

Angiotensin II 4

C. Commercial Angiotensin I and II: Analysis by

HPLC and Identification of the Impurities 5

D. The Analysis of Commercial Angiotensin I by TLC and

Electrophoresis 7

E. Angiotensin I Analysis for D-Amino Acids in the

Peptide 7

F. Angiotensin I Analysis for Non-Peptide Impurities 8

G. Angiotensin I - A Survey of UV Impurities in Six

Commercial Lots 10

H. Quantitation of the Peptide 13

Discussion 15

Specifications for Certification of an Angiotensin I SRM 19

Addendum - Angiotensin I Fluorescence Study

D. J. Reeder and L. T. Sniegoski 29

References 31
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Introduction

During the tenure of this contract, a series of

methods were evaluated and some were developed for

assessing peptide purity, particularly the purity of

Angiotensin I. Our objectives were: 1) the detection,

characterization and quantitation of impurities (peptide

and non-peptidc} in Angiotensin I samples; 2) the

selection of methods for peptide quantitation; and, 3)

the development of specifications and criteria by which a

peptide may be assessed for certification as a Standard

Reference Material (SRM)

.

Thin-layer chromatography (TLC) methods and high

performance liquid chromatography (HPLC) methods were

evaluated for use in separating the impurities from

Angiotensin I. Substances separated from Angiotensin I were analyzed for their amino acid content and composition

by the methods described in the attached reprints.

Unfractionated peptide was analyzed for D s tereoisomeric

amino acids by three methods. Total peptide was measured

by three methods. Non-peptide substances were studied

to determine their possible origin, their relative and

total proportion, their possible contribution to

absorbing components in the samples, and their physical

and chemical properties when these materials were

amenable to analysis.

1
Some of the results reported herein were obtained

with the cooperation of Drs . Jean Sealey (Cornell

Medical Center, N.Y., N.Y.)> Mahesh Khosla (Cleveland

Clinic, Cleveland, OH) and P. E. Hare (Carneige Institute, Washington, D.C.)*

Methods for Analysis of Peptide Purity

We have developed a high performance liquid chromatography (HPLC) method [2] that is capable of resolving

very closely related peptides. By modifying the solvent

system that was used in the method of Rivier [1]

.

Our

method is able to resolve such closely related peptides

as the [D-Pro]-, [D-Phe,]-, [alle]-, [D-Leu]-, and [Dg

asp, lie ]- analogs of Angiotensin I and provides an

important new means of determining the purity of Angiotensin I and Angiotensin II. The presence of the D amino acid

was confirmed by GC analysis of the peptide hydrolysate

on a chiral column [3]

.

In addition to resolving

closely related peptides, peptides characterized by

omissions and duplications of one or more amino acids,

can be resolved and characterized. We were not able to

test the resolution of the method for truncated "Angiotensin I peptides" or those with duplicate amino acids,

because the appropriate analog peptides are not available.

However, such analogs of Angiotensin II are available,

and they were tested.

2
A. Resolution of Peptides with Amino Acid Composition

Similar to Angiotensin I The capability of the HPLC for resolving peptide

products differing because of errors in peptide synthesis has not been exhaustively studied, but Table I illustrates the resolving capacity of the acetoni trile

triethylamine phosphate buffer system for peptides

closely related to Angiotensin I. Diasterioisomers of

Angiotensin I containing either D-Pro, D-Phe, or alle

can be separated from each other and from Angiotensin I (figure 1 and Table 1). Also [des Asp] [alle]- Angiotensin I can be resolved from [alle] -Angiotensin I.

[D-Phe] -Angiotens in I was resolved from a peptide

having 50 percent less aspartic acid. This aspartic

acid deficient peptide may be composed of either identical sequences on the a and 3 carboxyl groups of the Asp

or a sequence with duplication of each residue except

the N-terminal amino acid (figure 1, Table 1, peak 5

vs. 4). A similar error is seen in the comparison of

peaks 10 and 11. The resolution of [des Asp] , [lie 8]-

from [lie 8] -Angiotensin I suggests that this HPLC

technique can also separate the des [Asp] -nonapeptide

analog of Angiotensin I. The resolution of a group of

impurities from [D-Pro] -Angiotensin I having reduced

proportions of Phe and extra His and Arg demonstrates

3
that products resulting from extensive errors in peptide

sequence may be easily separated from Angiotensin I. [DLeu] -Antiotensin I (Figure 1 peaks 2 and 3) partially coeluted

with products having an erroneous composition; however, it

was adequately separated from Angiotensin I. Deviations in

the amino acid composition of these impurities demonstrate

the need for analyzing the amino acid composition of each

peak. These observations suggests that impurities having a retention time identical to that of Angiotensin I may exist;

however, our compositional analysis of the Angiotensin I peak suggest that none are present.

B. Resolution of Truncated Peptides Related to Angiotensin II

Des[Asp], Des[Asp, Arg] , and des[Asp, Arg, Val] Angiotensin II exhibit retention times which indicate that they

can be separated from each other and from Angiotensin II

(figure 2, Table II). Similarly, des [Phe] -Val^-Angiotensin

II was separated from [Val ^

] -Angiotens in II. These samples

also contained at least one additional peptide which exhibited

either a duplication of an amino acid (His in peak 11) or

truncation of the peptide (particularly the Tyr in peaks 3

and 6 and possibly in peak 11 as well as His in peak 9) . By

HPLC it was also possible to separate [Val ^

] -Angiotens in

isomers that differed in one amide linkage, i.e., of the Asp

by the amide bond of either the a or 6 carboxyl group to the

a amino group of the Arg (Figure 3). Furthermore, the 6 Asp

4
isomer of [val^] Angiotensin II contained a small amount of

the a isomer.

C. Commerical Angiotensin I and II: Analysis by HPLC and

Identification of the Impurities

The purity of commercial Angiotensins I and II was

examined by our HPLC method. The results of these studies

are reported in the appended reprints. Briefly, Angiotensin

I and II contain one peptide impurity each. Each impurity

appears to form during storage of the peptides. Angiotensin

I was found to contain also a trace component, detectable

only by radioimmunoassay (RIA) (Table III Fraction 12-14).

The trace RIA-positive material cochromatographed with 3

ultraviolet (UV) -positive (254 nm) components which were

peptide-free bv amino acid analysis. These UV positive components (12, 13, 15 min. ret. time) were not present in crude

Angiotensin I; therefore, we believe, they were introduced

during the purification step and are not related to the RIA

positive material (see appended nrenrint) . Four additional

non-pentide components with absorbance at 254 nm were detected

however, thev were found only after the aqueous solution of

the peptide was allowed to come into contact with the

rubber stopper of the vial. The presence of HPLC peaks due

to these materials could be avoided bv not exposing the

peptide solution to the stopper. The chemical composition

of the other non-pentide components was not pursued when it

was found that they evaporated with the aqueous solvent at

100 °C under a stream of nitrogen.

5
The peptide impurity in commercial Angiotensin I had

the same amino acid composition as Angiotensin I (Table IV)

An analogous result was found for the peptide impuritv in

Angiotensin II (Table V)

Ansiotensin I and the peptide impuritv were separated

on the octadecyls ilane column and collected. Each sample

was dried under N

2 at 100 °C, dissolved in a neutral bicarbonate buffer and tested in a oressor assay on rats by Dr.

M. Khosla. In the biological assav, the peptide impurity

from the Angiotensin I orenaration exhibited anproximately

30 percent of the expected specific activity and the

Angiotensin I exhibited approximately 20 percent of the

expected biological specific activity thus indicating that

the materials eluted from both peaks were biologically

active peptides. The loss of biological activity probably

occured during the concentration of the samples prior to

bioassay. This biological active impurity was found in

Angiotensin I that had been stored in its original dry form

for more than one month at 4 °C It was not detected in

either of two different lots of Angiotensin I from Beckman

obtained 9 months anart when they were analyzed immediately

unon receipt. (The samnles were examined on the same column).

For fresh and stored Angiotensin I the recoverv of the Angiotensin T in the HPLC exneriments was 95 percent of the total amount

of neptide ap^nlied to the column ba'^ed on the amount of pentide

6
stated on the label, when the recovery was deterniTne^

either by amino acid analysis or by RIA. Furthermore, the

Angiotensin I measured by RIA in the collected chromatographed peaks was equivalent to that measured in the unfractionated material, indicating that the fractionated impurities

which are not included in the weight stated on the label do

not modify the RIA method of analysis.

D. The Analysis of Commerical Angiotensin I by TLC and

Electrophoresis

The lack of peptide impurities in commercial Angiotensin I

is consistent with the general findings obtained by TLC

(Figure 4). With the possible exception of a peptide impurity,

evident as a small extended tail which was barely detectable

on cellulose using n-butanol, pyridine, water (65:35:35) as

the solvent and the Pauly reagent for detection, there was

no other evidence of peptide impurities.

Only a single spot was observed on cellulose using nbutanol, acetic acid, water (4:1:5); n-butanol, pyridine,

water (65:35:65) and n-butanol, acetic acid, water, pyridine

(15:3:12:10)

.

A single peptide impurity which migrated slightly

slower than Angiotensin I was detected by electrophoresis at

pH 8.6. A single spot was observed at pH 2.5.

E. Angiotensin I Analysis for D-Amino Acids in the Peptide

The presence of "Angiotensin I peptides" containing

D amino acids should be of serious concern because these

- 7 -
types of dias tereomers differ in biological activity from

Angiotensin I. Several such dias ter iomers of Angiotensin I

can be separated from Angiotensin I by HPLC (section A)

Kohsla has demonstrated that [D-His ] -angiotensin I is

separable from the [L-His ] -diasteriomer by TLC on cellulose

in n-butanol, pyridine, water (65:35:35) [5]. Frank et al [3]

demonstrated that chromatography of derivatives of hydrolysates of peptides on a chiral column can provide a rapid

quantitative method for the separation and measurement of

the D § L amino acids. The examination of Peninsular and

Beckman Angiotensins by TLC revealed no detectable [D-His]

Angiotensin I (Figure 5) (interpretation of M. Khosla)

Also, examination of the 2-OH propyl TFA derivatives of the

amino acids from hydrolysates of new and aged Beckman

Angiotensin I did not reveal significant amounts of D amino

acids (i.e., greater than 1%) above the amount generated by

the racemization that occurs during hydrolysis. This method

was not useful for the analysis of D and L His or Arg. We

expect to use a method being developed by E. Gil-Av for

these amino acids. Thus three independent methods failed to

reveal the presence of diasteriomers

F. Angiotensin I Analysis for Non-Peptide Impurities

Analyses for non-peptide impurities were performed by

three independent methods: 1) detection of HPLC peaks

absorbing UV light at 254 and 280 nm; 2) mass spectrometric

8
analysis, using a programmed volatilization for materials

that evaporate in the mass spectrometer between 30 and 300

°C; 3) amino acid analysis by the method of standard additions. The UV absorbing non-peptide impurities which are

described in section C and the appended reprint arise from

two sources; a group of three HPLC peaks eluting before

Angiotensin I that appear to be introduced during the

purification steps, and a group of four HPLC peaks eluting

after Angiotensin I that are from the rubber stoppers.

The mass spectrometric analysis indicated the presence

of dimethyl siloxane polymers, triethylamine , acetic acid,

and a compound which decomposes above 300 °C with the

release of benzyl alcohol. The first three of these compounds absorb little or no UV light and thus are not detected by simply monitoring the UV absorbance at 254 nm or

280 nm.

The total amount of impurity in the Angiotensin I was

determined by a standard addition experiment. Lyophilized

Angiotensin I was weighed and dissolved in distilled water.

Phe was weighed and dissolved in 0.1 mol/L KOH in a volumetric flask. The concentration of this solution was

confirmed by measuring its absorbance at 258 nm and calculating its concentration by use of the published molar

extinction coefficient 209.6. Phe was added to aliquots of

an Angiotensin I solution in amounts equal to 0, 1, 2, 3 and

4 times the approximate content. The 5 solutions were dried

9
in a stream of nitrogen, hydrolyzed for 20 min in 6 mol/L

HCl at 155 °C, dried again, taken up in a suitable amount of

solvent and analyzed on the amino acid analyzer. The Phe

content of each sample was normalized against each of four

amino acids in the Angiotensin I hydrolysate and then plotted against the calculated amount of Phe added to each

sample. The data were analyzed by linear regression and the

amount of Phe present in the Angiotensin I was determined

from the intercept of the line with the x axis (concentration scale) . From this number the Angiotensin I content was

calculated and compared to the weight of Angiotensin I that

had been added to each sample. The results of this calculation indicate that 5 percent of the weight of the Angiotensin

I was non-peptide (Table VI). This value was found to be

independent of the amino acid used to normalize the Phe

concentrations. This 5 percent non-peptide impurity value

includes those non-peptide impurities detected by HPLC and

MS.

G. Angiotensin I — A survey of UV Impurities in Six Commerical

Lots

To accomplish the objective of surveying the purity of

commercial Angiotensin I, samples from various lots obtained

from several commercial sources were dissolved in water

to a final concentration of 1 mg/mL (based upon the

suppliers stated peptide content) . Thus these results

reflect both the peptide impurities and non-peptide

10
impurities that absorb at 254, 280, or 220 nm and

inaccuracies and variation in peptide content between

vials and between lots. The impurities detected at 254

nm also absorbed at 280 and 220 nm. The non-peptide

impurities exhibited greater absorbance at 280 nm while

the peptides exhibited greater absorbance at 220 nm.

we evaluated the purity of 6 lots of Angiotensin

I from three different primary sources. One to three

vials were examined from each lot. Each value is the

average of duplicate measurements. We attempted to

isolate and identify the impurities but were unsuccessful in our attempts to concentrate them after they were

collected. Therefore, the peak areas reflect only a

concentration relative to the total absorbance, since

the chemical structure and molar absorbtivity of the

material in these peaks is unknown.

Two independent HPLC systems of analysis were

used. One system employed an aminosilane column (Waters

Associates) with 0.001 mol/L acetic acid imethanol , 3:7

(V/V) as the mobile phase and the other system utilized an

octadecylsilane column (DuPont Zorbax ODS) with 0.1

mol/L triethylamine phosphate (pH 3 . 5) : acetonitrile

,

81:19 (V/V) as the mobile phase. Table VII summarizes

the relative peak areas of the Angiotensin peak and the

two non-peptide peaks that were resolved by use of the

aminosilane column. The three peaks were analyzed separately

11
for their amino acid composition to determine the

amount and nature of the peptide. They were analyzed

also by RIA for Angiotensin I by Dr. Jean Sealey. Both

the RIA and amino acid analyses indicated that 100 percent

(within 2%) of the original peptide was recovered after

chromatography in the peak labeled Angiotensin I. No

peptide was detected the solvent front.

A comparison of the data observed for the samples

which were examined indicate that the relative amount

of the non-peptide impurity varies from vial to vial in

a given lot, (e.g., Lots lA and IB) and from lot to lot

(e.g., lot lA vs lot 2A) . The cleanest material appears to

be the lot obtained from Source 2 (the Protein Research

Foundation) . In addition to the variations in the content

of non-peptide peaks, the amount of peptide varies from lot

to lot - from 256 ± 11 (Lot 3A) to 328 ± 12 (Lot 3C) in

relative area units, i.e., a difference of 72 units or

28 percent of the lower value.

The material fractionated by the second method on

an octadecyls ilane column was also analyzed for peptide

composition, RIA-detectable material and biological

activity. As mentioned previously, the non-peptide

peaks contained approximately 0.3 percent of the total

RIA-detectable material. The peaks at 12, 13 and 15

min in Table VIII contained no detectable peptide by

amino acid analysis. The magnitudes of these peaks

12
varied in a manner that paralleled the inter-lot and

inter-vial variation of the non-peptide peaks listed in

Table VII. All of the Angiotensin I samples that were

examined in this survey were at least 3 months old.

These samples also fractionated into 2 peptide-containing peaks (retention times 20 and 25 min) each of which

had the amino acid composition of Angiotensin I and

exhibited pressor activity in the rat assay. The peak

at 25 min was equivalent to 9-13 percent of the area of the

peak at 20 min. The relative magnitude of these peaks

showed little intervial and interlot variation. The peak

at 25 min was not detected in samples that were examined

within 1-3 weeks after receipt. On the other hand, the

actual area of the two peaks did exhibit inter-vial and

inter-lot variation similar to the variation observed in

the area of the peptide peak when the Angiotensin I was

analyzed on the aminosilane column (Table VII vs. Table

VIII) .

H. Quantitation of the Peptide

All peptide samples were disolved in distilled water

at a concentration of 1 mg/mL based upon the weight stated

on the vial. Measurement of the areas of the peaks

from HPLC of different lots of Angiotensin I indicated

a large inter-lot variability in peptide content (see

section G) . A smaller variability was found in the

13
inter-vial peptide content. Results for Peninsular

.Angiotensin I (Lot 3A) based upon amino acid analysis

of the collected HPLC peak indicated that this preparation contained between 95 and 97 ug per 100 mL when the

total contents of the vial were dissolved in water to a final concentration of 1 mg/mL (the mass of peptide was

based on the value given on the label) . Thus samples

with peak areas greater than or less than those for source 3

sample .A contain more or less than the peptide content

stated on their label.

The standard addition analysis provides an independent measure of the total peptide content (see section

F) , but has the disadvantage of being extremely timeconsuming. The phthalaldehyde method [6] for measuring

total peptide was evaluated (see addendum) and was found

to be useful for the quantitative measurement of peptide

content in the range of 12-120 ug/mL, and is thus adequate

for reference samples containing 0.5 to 1.0 mg of peptide.

This method is less time-consuming than the standard addition analysis. For peptide homogeneity studies, we believe

that the standard addition analysis should be used to calibrate the peak area and the fluorometric assay methods

if they are used as the analytical techniques for measuring

the peptide and non-peptide content of the samples.

14
Discussion

An HPLC method has been developed for the analysis

of Angiotensin I. This method is capable of resolving

Angiotensin I from other peptides, such as D-amino acid

diasteriomers and peptides resulting from errors in the

Angiotensin I synthesis. des[Asp]-, des[Asp, Arg]-, and

des[Asp, Arg, Val] -Angiotensin II and their impurities

(except for peak 6} can be resolved from Angiotensin II by

use of the HPLC method. [3-Asp, Val^] -Angiotensin II can be

distinguished from [a-Asp, Val^ ] -Angiotensin II and from

des [Asp] -Angiotensin II; des[Asp, a-Ile^] -Angiotensin I can

be separated from [a- lie ] -Angiotens in I. Thus, this system

is capable of resolving the impurities which are of primary

concern in the analysis of Angiotensin I. The presence or

absence of D-dias teriomers can be determined by analysis for

most of the D-amino acids using gas chromatography. The

levels of D-Asp and D-Phe which were detected are typical of

the levels generated during the hydrolysis procedure due to

racemization.

Newly synthesized Angiotensin I chromatographs as a single peptide, showing one peak having the amino acid

composition characteristic of Angiotensin I. HPLC of aged

Angiotensin I (stored as received for 2-12 months at 4 °C)

reveals 2 peptide peaks having identical amino acid compositions. The newly evident peak comprises 9-12 percent of

the total peptide. Since this peptide has the requisite

15
amount of aspartic acid, the peptide cannot be des[Asp]-

Angiotensin I. The following experimental facts suggest

that it is [3-Asp] -Angiotensin I.

1. A significant peptide impurity is found on

electrophoresis of Angiotensin I at pH 8.6. It migrates

somewhat less rapidly than the major component. The

impurity is not observed at pH 2.5. This observation

is consistent with the resolution of [3 -Asp] -Angiotensin I under slightly basic conditions [4]

2. A peptide impurity is found by TLC on cellulose with butanol, pyridine and water (65:35:35 V/V)

which is consistent with the resolution of [3-Asp]

Angiotensin I under slightly basic conditions.

3. The greater retention time of this peptide

impurity on an ODS column using TEAP : acetonitrile is

consistent with the increased retention time found for

[3-Asp, Val^ ] -Angiotensin II as compared to the a isomer

4. The presence of acetic acid in the lyophilized

material would provide, in the presence of moisture, an

appropriate concentrated acidic environment necessary

for the catalysis of the a-3 rearrangement of the Asp

amide bond with the a amino group of the Arg.

5. The specific activity of the impurity as

measured by bioassay was found to be 50 percent greater

than that of the major peptide - a characteristic of 3 Asp

16
Angiotensin I [4]

.

Thus the evidence strongly supports

the formation of 6 Asp Angiotensin I during aging.

The non-peptide impurities do not appear to significantly affect the Angiotensin I measurement by RIA.

Their effects on the biological assay have not been

assessed. The non-peptide impurities detected by HPLC

appear to be present in only small proportions in two

of the lots evaluated. Impurities derived from the

stopper can be obviated by avoiding contact between the

Angiotensin solution and the stopper. The dimethyl

siloxane polymers that we found probably arise from the

silanizing process used on the glass to minimize absorption of the peptide and therefore may not be avoidable.

The total non-peptide impurities can be assessed by the

standard addition method. The non-peptide impurities

comprise approximately 5 percent of the dry weight of the

peptide sample for the samples we analyzed.

The inter-vial homogeneity of peptide content can

be measured by three methods: the standard addition

m.ethod to obtain the absolute peptide content, the HPLC

area measurement method, and the o^-phthalaldehyde method.

The last two methods can yield information only on the

relative homogeneity, unless they are normalized measurements by the standard addition method which provides a more

absolute basis for the comparative measurements.

17
Conclusion

The primary problem in the preparation of an

Angiotensin I standard appears to be the spontaneous a

to 3 rearrangement of the amide linkage of the carboxyl

group of the aspartic acid residue. This rearrangement

results in an enhanced biological activity of the

Angiotensin I preparation in proportion to the content

of the rearranged product. Since it is 50 percent more

active than Angiotensin I, if present as 30 percent of the

preparation, then samples would show 15 percent higher

activity in the bioassay. However, RIA does not distinguish between the two forms (personal communication

from Dr. J. Sealey) ; hence, activity by RIA would be

unchanged regardless of the ratio of these isomers.

The impurities present in the Angiotensin I do not

appear to modify the sensitivity of the RIA assay for

Angiotensin I. Thus if the peptide content is accurately

stated then the peptides we have analyzed appear to be

suitable for use as RIA standards, but not as standards

for biological assay unless their content of [3-Asp]

Angiotensin I is periodically assessed. With this

stipulation. Angiotensin I now available can be certified as a standard.

18
Specifications for Certification of an Angiotensin I SR^l

These criteria are based on the studies summarized

herein.

1. When examined by HPLC on a Zorbax ODS column

using TEAP buffer 0.1 mol/L, pH 3.4: acetonitrile

(81:19 v/v and 75:25 v/v)

,

the area of all non-peptide

peaks should be less than 1 percent of the area of the

Angiotensin I peak. Samples should be examined separately at each of the solvent ratios.

2. Upon receipt, the peptide should be free of

peptide impurities a) when examined by HPLC analysis as

described in paragraph 1 of these specifications; b)

when examined by TLC on cellulose using each of the

following solvent systems:

Butanol, acetic acid, water (BAW) 4:1:5 v/v

Butanol, pyridine, water (BPW) 65:35:65 v/v

Butanol, pyridine, water (BBW) 65:35:35 v/v

Butanol, acetic acid, water, pyridine (BAWP)

15:3:12:10 v/v

and c) when examined by electrophoresis at pH 2.5 and

pH 8.6.

3. The amino acid composition should be within

±3 percent of the theoretical composition for each

amino acid.

19
4. The peptide should contain less than 1 percent of

any [D] amino acid when examined by GC analysis on a chiral

column [3] or by the new method of E. Gil Av (personal

communication)

5. Total non-peptide material should be less than 5

percent when estimated by the standard addition method using

Phe as the standard.

6. Each vial should contain between 500 and 525 yg of

peptide

20
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cti H

cr> CNl LT) vO cr> CNl 00 rH

rH o o 00 CNl o o LO

rH • • o • • • O • • • • rH rH rH CNl rH rH 00

LO CNl CNl rH CNl o CNl LO

O LO C7^ C7i o Ol CNl CD o • • o • • • O • • • • rH CNl H" o X Ol CNl LO [>. to rH o cn 00 O o CD tH 00 rH CD rg

<3^ • • • rH rH rH rH rH rH

(N1

7)

w 0 Pi H" LO o LO 00

O

cd 130 o o 00 o o 00 rH CD CD CD CNl •H 0 p p Ph 0 rH rH rH rH 00 a X rH 0 u s CNI CNl CNl LO CD CNl to LO to

a, •iH 0 o CT> o 00 o tH o CD p:

T3

a rH rH tH CNl rH to •H cd P5 u P cd < bO 0 o o H" o X o o LO O Ph o rH 00 o CO X LO o CTl o P MP

e: cd rH rH o rH CNl H" CNl •H £ s o < pX X X C7l o to to CD to X Q u Ln CO cn 00 rH rH rH O rH O

M-i

p rH rH rH CNl rH rH CX5

O o CNl p

t/1 t>~ o LO o CO LO IN- o CD •H 7) H“ H- rH 00 o CD o o o <D LO

in o

>> •rH rH rH rH CNl rH

rH

p CNl ci cd S Pi rH 00 K5 rH CNl rH to LO CNl rH < K) o 00 cn o LO LO rH rH o rH Td oij •H CNI rH rH rH to tH rH o

•H

u rH

u < < o CTi 00 rH CNl to LO o to o c Cd CX3 cn 00 rH CD rH rH o o CNl rn •H rH

•H s rH rH CNl rH rH C-- S< U<oc

•H6<

Pu rH 0p 0 0 P 0 71 bO

O

71 05p P 0 P P P < > cd p X E- X < PLh

•H0e

*HH0

ctJ

21

Angiotens

in
1—

m

X in

oo rH CM CM

C c o • • • • c + • •H rH CM rH

t/5 rH c

(U Xo rH H- rt rH • H CO o o o

00 o c o • • • • o + c i-H rH rH rH CTi <

<x K3 CN] H" LO cn o CO CTl o CM oo o • • • • • c +

to CO rH tH CO s rH

f-l o rH CM iH rH

tin rH o cy> o Oi o • • • • • o +

t3 00 rH rH rH

m

0) rH X

03

X

m 00 00

u o 00 c o in p o • o • • • o + • p rH rH rH LO

fH H

<X rH LO in o O CO CO Ln o o o 00 u o • to X o rH rH rH + CM e rH

o3

p

f-t

p rH rH uo in CT> o

Ofl

X CO CO rH o o oo o € u c • (U X p LD rH rH rH + CO

!—

03 2

XI s

03 o X in i_nm •H* inm

U P o o CM rH ooX

cu

X o • U Ph X rH rH rH rH o + CM

rH

CD X C<1 o CM 00 X CO CO Oi CM O 00 oo X o • • • • • • • £ Ln rH rH o + C7l o

!h X 00

m oo UO o rH oo Oi o X

to • • o • o • • • X rH CNl rH + in

03O

OhX csi rH o o oo LO rH CT> LO

tn • • o • o • • • to rH rH o rH o + CM •H

to K) rH CO in rH LD

1—

CO o CO oo rH o o O

o3 CNl p rH rH rH rH rH LO < rH T3 CTi o m rH CO 00 o

•H

p CT> o 00 00 rH CO CO o CM

u rH < rH rH rH CM

rH o /—

p • •H . (D P s a rH p p tU p bO o X E -H < to p rH X X • iH P P <u *H E < > hH X Ph X < Ph Pi X M_

+->

CD

+-> U

<U

+-> (D

cd

:sopo5-. • &, <u

+J

rt cd X -H

M-i P to X

•H P o p P

to P X

p P o X p P P u cr X p o •H o X r-i X

•H Ph Ph

p o

bO

g S •H

p P P P < cn CO + P X u - 22 - i
Table III

Radioimmunoassay of Angiotensin (100 yg) Fractionated

on a C^g Column Using TEAP : Acetonitrile Solvent

Fraction Time

Interval (Min)

Ng

Angiotensin I 0-2 23

2-4 17

4-6 14

6-8 20

8-10 22

10-12 16

12-14 140

14-16 89

16-18 79

18-20 53800

20-22 41500

22-24 364

24-26 245

26-28 188

28-30 131

30-32 98

Assay performed by Dr. J. Sealey

•u Solvent flow rate ImL/min.

23
Table IV

Amino Acid Ratios for Angiotensin I Peaks

Retention Time

Amino Acid 20 Min^ 2 5 Min

Asp .87 .90

Val .99 1.06

He .88 .88

Leu 1.11 1.13

Tyr .92 .89

Phe 1.11 1.13

His 2.08 2.15

Arg 1.05 1.00

Pro 1.03 1.01

yg Peptide 45.9 3.3

Recovered

Solvent 811 TEAP : 19% Acetonitrile

^Angiotensin I

24

o\o
Table V

Amino Acid Ratios for Angiotensin II Peaks

Retention Time

Amino Acid 12.2 Hin^ 15.7 Min

Asp . 99 .93

Val 1.00 1.07

lie .87 .91

Tyr . 83 .89

Phe 1.11 1.13

His .98 1.01

Arg . 98 1.07

Pro 1.00 1.05

• yg Peptide

Recovered 32.7 yg 1.00 yg 86 % Solvent 82.5% TEAP : 17.5% Acetonitrile

Angiotensin II

Recovery

25
Table VI

Purity of Angiotensin I by Standard Addition

I Added Phe Added Peak Ht

Phe

yg nmol Peak Ht

TaT

26.67 0 0.207

26.67 17.52 0.410

26.67 35.05 0.569

26.67 52.52 0.783

26.67 70.09 0.959

linear regression

X Intercept 19.

analysis R = 0.999

53 n mol = 25.33 yg protein

% Purity = X 100 = 94.98%

26
Table VII

Magnitude o£ Impurity Peaks in Angiotensin I (AI)

Relative Peak Areas

Source Lot

of Impurities

(AI = 1000)

Actual Peak

Area AI

1 A 175 0 299

280 18 286

269 30 296

B 37 3 312

2 A 15 34 262

0 0 265

3 A 41 4 267

129 43 256

132 19 143

B 53 15 323

• 54 19 316

C 24 19 318

Analysis conditions:: NH^ column 0.001 mol/L acetic acid:

methanol '5:7 V/V. All peptide samples were dissolved in

water just before analysis at a concentration of 1 mg/mL

based on the peptide weight stated on the label.

27
Table VIII

Magnitude of Impurity Peaks in Angiotensin I (AI)

Relative Peak Area

Retention

(AI = 1000)

T ime

Actual Peak

Area

(Ret. time

20 min.)

Source Lot 12 Min 13 Min 15 Min 25 Min AI

1 A 20 159 61 133 540

17 247 81 116 580

10 108 65 105 611

B 2 52 19 121 630

2 A 1 9 1 120 548

1 1 1 128 550

3 A 2 69 19 118 566

4 130 37 103 563

9 100 27 116 558

B 5 74 23 92 739

5 69 21 116 773

C 6 74 16 102 724

7 64 16 103 685

Analysis Conditions: ODS Column 0 . IM TEAP pH 3 . 5 : acetonitrile

81:19 V/V.

All peptide samples were dissolved in water at the time of

analysis at a concentration of 1 mg/mL based upon the weight

stated on the label.

n n
Addendum

Angiotensin I Fluorescence Study

D. Reeder and L. Sniegoski

Several conclusions may be reached as a result of this

study: (1) Angiotensin I may be analyzed for homogeneity in

its packaged form of 0.5 mg/vial, provided there are no

extraneous amino acids. Adequate sensitivity is achieved by

the fluorescence method in the 12-120 yg/mL range (4-40

yg/mL in final detection system) . A suggested procedure is

to reconstitute the contents of the 0.5 mg vial with 750 yL

water, ad analyze 4 x 120 yL samples from each vial; (2)

the sensitivity of the orthophthalaldehyde reagent for the

peptide is about 10-fold less than that for the amino acids.

This suggests the reagent is only reacting with the terminal

amino acid; (3) Angiotensin II reacts slightly better in the

fluorescence assay system than Angiotensin I; (4) standard

curves for the individual amino acids may be generated to

give correlation coefficients greater than 0.99. Proline

does not react in this system. On a molar basis, tryosine

is about 1/2 as reactive, and valine about 2/3 as reactive

as nor-leucine. All other aTnine acids ^’re ^^pp'^ox'’ ma^’^el"'’’ equal in reactivity, although isoleucine gives the highest

readings

.

29
Corrected

ymoles/

liter

in

Final

Solution • <4-1 O' to 'St rH 04 o- O' o U 0) cn cn CTi C7> 00 04 04 04 O' O O > o> C7> CT> cn cr> 04 rH 04 04 U U o o O o o O O o O o t u

0) -t-> LO 00 o rH OO o O' LO

+-> oo to vO o vO 04 04 04 vO to C OJ LO 04 'St to rH o 00 LO VO

t-H

O • o 04 rH 04 to rH rH LO

0) vO rH 04 to rH to vO o oo o CT> VO 04 04 VO rH LO 04 o S vO C7> vO vO t^ rH 04 vO

CO (-H rH rH 04 rH rH 04 o vO 04

rH rH rH rH rH rH rH 1

CT) CT> O'. o 00 o vO 04 LO

rH rH rH rH 04 04 04 rH rH rH

to to to to to to 04 to to to

tin • o o o o o o o o o o

CTi rH LO 04 04 LO 04 o

K) to to 04 LO to 04 04 04 to o vO vO VO vO VO LO VO VO VO

M • o o O

O o O o OO o

LO o^ 00 rH rH OO LO 04 00 rH O' VO 'St rH O' OO 'St LO vO

CM 04 04 04 to 04 rH 04 04 04

Q rH rH rH rH rH rH rH rH rH rH

vO to to to- vO 04 to LO rH

00 CT> vO o 04 VO 04 LO rH rH rH rH 04 rH 04 rH t—H rH

U to to to to to to 04 to to to

to VO oo LO 'St 04 'St VO 04 to 00 to o LO 04 04 00 o to to to 04 LO to 04 04 04 to

pq VO VO VO vO vO VO LO vO VO VO

vO to vO rH OO 04 04 00 LO 0^ O' rH O 00 'St 04 O' o < vO rH t^ CO 'St LO vO

CSl 04 04 04 to 04 rH 04 04 04

rH rH rH rH rH rH rH rH rH rH

'c^

0)9 00 to UO vO LO LO 04 rH O' 04 S 9

in vO 'St CTi 04 VO 04 rH 04 to ^ S to to to 04 'St to rH to to to S =!- 04 04 04 04 04 04 04 04 04 C4

1—1 +j T3

aJ

C CD 00 LO o D 3 X vO to rH to 04 o O' to M O C>0 • • • rH 00 o 04 VO rH to y E -H CJi rH VO ^ to to o rH rH vO vO 04 C'- :s to to to to 04 'St 04

LO

O CNI vj LO cn o LO LO LO o O o o O rH O' o^ cr> to to to CNJ vO 04 rH

rH rH •\ 5h Q 00 o LO o o o oo vO rH O' bO o S to to to to to to 04 04 E M-l 3. O'o rH 04 04 04 04 04 rH 04 04 o to LO rH rH rH LO LO rH O'

rH to LO to to to vO rH 00 rH

04 rH rH rH rH rH rH rH rH rH

o3 00 cu V) o 3 u rj o u rH •H

•-r^

c/) HH

0) o2 <-e- Oh :>

30
References

1. Rivier, J. E., J. Liq . Chromatogr . , ^:343 (1978).

2. Margolis, S. A., and Longenbach, P. J. , HRC § C£ in press

(1979) .

3. Frank, H. , Nicholson, G. J. and Bayer, E. , J. Chromatogr .

Sci . , 15_:174 (1977) .

4. Rohiker, B., and Schwijzer, R. , Helv . Chim Acta , 47:2357

(1964) .

5. Khosla, M. C., Bumpus , F. M. , Hagashi, Y., Nakajima, T.

,

Watanabe, T.X. and Sokabe, H. , J. Med . Chem. , 20:315 (1977).

6. Reeder, D. J. , Sniegoski, L. T., and Schaffer, R. , Anal . Biochem. 86:490 (1978).

31
Reprints

1. S. A. Margolis and P. J. Longenbach, "Analysis o£ Purity of

Commercial Peptides by High Resolution Liquid Chromatography"

in The Role of Peptides in Neuronal Function , Eds. J. Barker

and T . Smith , Marcel Dekker (in press)

2. S. A. Margolis and P. J. Longenbach, J. High Resolucion

Chromatogr. and Chromatogr. Commun., 2:255(1979)
Figure Captions

Figure 1. Chromatograms of D-Diasteriomers of Angiotensin I.

The peptides were dissolved in water at a concentration

of 1 mg/mL. 50 yg was applied to an ODS column that

was equibrated with 0.1 M triethanolamine phosphate,

pH 3.5 (TEAP) : acetonitrile 81:19 v/v at a flow rate of

1 mL/min. The fractions absorbing at 254 nm were

collected and analyzed for amino acid content (Table

1). The D peptides were a gift of Dr. Susan Oparel

.

Figure 2. Chromatograms of truncated form of Angiotensin

II. The peptides were dissolved in water at a concentration of 1 mg/mL. 50 yg was applied to an ODS

column that was equilibrated with TEAP buffer: acetonitrile (81:19 v/v) at a flow rate of 1.25 mL/min. The

fractions absorbing at 254 nm were collected and

analyzed for amino acid content (Table II)

.

Figure 3. Chromatogram of a and B Asp Val^ Angiotensin I The peptides were dissolved in water at a concentration

of 1 mg/mL. 50 yg was applied to an ODS column that

was equilibrated with TEAP buffer : acetonitrile (85:15

v/v) at a flow rate of 1 mL/min. The fractions absorbing

at 280 nm were collected and analyzed for amino acid

content. The 3-asp Val^ Angiotensin II was a gift

of Dr. Michael Peach.

33
Figure 4. Thin layer chromatograms o£ Angiotensin I samples

The chromatograms were prepared by Dr. M.

Khosla [5] using cellulose plates and the solvent

systems indicated on the chromatograms.

Figure 5. Electrophoretic chromatograms of Angiotensin

I samples. The chromatograms were prepared by Dr.

M. Khosla using the standard methods in his

laboratory at the pH’s indicated on the chromatograms [5] .
TIME (min)
des (Asp Arg Val) All
/3Asp

Val®

All

TIME

(min)
ic*r
2t£C '7S

^£CKMfih/ TC P£NfN^ULA

i ^

2>R M- C^ KHoSLfii

Short Communications 10092

Separation of Structually Similar, Bioiogicaily Active Peptides from

Their Impurities

S. A. Margolis* and P. J. Longenbach

National Measurement Laboratory, National Bureau of Standards, Washington, DC 20234, USA.

Key Words:

Liquid Chromatography, HPLC

Peptide analysis

Angiotensin I and n, peptide hormones, resolution of impurities

Interest in the use of high resolution chromatography for the separation and analysis of peptides has grown considerably

during the past few years [1-5]. The purity of biologically acitve peptides is of particular importance for meaningful studies of

their pharmacological mechanism of action and use in humans.

A simple isocratic method that provides ample resolution of very

closely related peptides as well as resolution of many nonpeptide impurities has been developed by modifying the work

originally reported by Rivier [5]. The peptides which were studied are listed in Table 1. They were

either purchased from commercial sources or were supplied as

gifts from Dr. J. Barker (National Institute of Neurological and

Communicative Diseases and Stroke) and Dr. M. Khosla (Cleveland Clinic). Analyses were performed with a commercial chromatographic system on an octadecylsilane column. The solvents were composed by volume of either 1 7.5, 19, or 25% acetonitrile (glass distilled) and 0.1 mol/l triethylamine phosphate buffer, pH

3.5 (TEAR). Eluted peptide-containing peaks were identified by

amino acid analysis.

This method is capable of separating the decapeptide angiotensin

I from the octapeptide angiotensin II, resolving [ile5]-angiotensin

II from [va|5]-angiotension II (which differ only by a methylene

group), and met enkephalin from leu enkephalin.

By 254 nm monitoring of preparations of individual peptides, several chromatographic peaks were resolved (e.g., see Rgure 1 for Angiotensin I). Different preparations of the same peptide were found to contain different groups of impurities (Table 1) and

different proportions of the same impurity. The same peptide

preparations revealed different impurities depending upon the

solvent system employed (Figure 1).

Preliminary analysis of our data indicates that some peaks were

non-peptide impurities from impure peptide; others were intro- duced during the purification; and some were contaminants from

the serum stopper. Several peptide preparations were found to contain several percent of peptide impurities, for example, des

[ile5]-angiotensin II in angiotensin II.

Table 1

Values of K' for Several Biologically Active Peptides and the Number of Other Peaks Detected at A2S4-

Solvent composition a 17.5% 19% 25%

K' other peaks K' other peaks K' other peaks

Peptide

Somatostatin b b 8.5 5 Substance P b b 3.0 5 Angiotensin I b 16.0 9 1.7 6

Eiedoisin 8.0 5 6.4 5 <1.0

[Leu]-Enkephalin (lot A) 11.4 5 6.2 5 <1.0

[Leu]-Enkephalin (lot B) 11.5 6 6.2 5 <1.0

[lle5]-Angiotensin II 7.0 6 4.1 3 1.0 3

[Met]-Enkephalin (lot C) 5.4 7 3.2 8 0.9 5

[Met]-Enkephalin (lot D) 5.4 9 3.0 5 <1.0 LRH 4.5 1 2.6 1 <1.0 3

[Val5]-Angiotensin II 3.0 1 2.1 5 <1.0

(a) percent acetonitrile (by vol.) in 0.1 mol/l triethylamine phosphate (pH 3.5)

(b) not measurable

Journal of High Resolution Chromatography & Chromatography Communications VOL 2, MAY 1979 255
Short Communications 10092/10093

Resolution of impurities in Angiotensin I by different solvents. A 25

|jg sample of angiotensin I dissolved in the eluting solvent was applied to the column and eluted isocratically with either acetonitrile: TEAR buffer 19 : 81 (A) or 25 : 75 (B). All eluted peaks were collected and analyzed

for amino acid composition. Only the angiotensin I peak (AI) contained amino acids and these were in the correct stoichiometric ratio for AI. 94 percent of the AI was recovered. Detection was at 254 nm at 0.02 AUFS.

Investigators using commercially prepared peptides for pharmacological and physiological studies should be aware of these

impurities and their possible effect on their experiments.

Acknowledgement

This research was partially supported by a research agreement with the National Heart, Lung and Blood Institute. National Institudes of Health, United States Public Health Service.

References

[1] I. Molnar and C. Horvath, J. Chromatogr., 142 (1977) 623.

[2] K. Krummen and R. Frei, J. Chromatogr., 1 32 (1977) 27.

[3] M. Takai, S. Kumagaye, T. Kishida and S. Sakakibara, Proc. 16 Symposium on Peptide Chemistry, in press.

[4] J. Feldman, M. Cohn and D. Blair, J. Liq. Chromatogr., 1 (1978) 833.

[5] J. Rivier, J. Liq. Chromatogr., 1 (1978) 343.

MS received: 19 March 1979

Post-Column Derivatization Using HPLC Cassettes 1 0093

E. Oelrich* and D. Theuerkauf

E. Merck, Department for Pharmaceutical Analysis and Quality Ck)ntrol, D-61 00 Darmstadt, Germany

Key Words:

Liquid Chromatography, HPLC

Detection by post-column derivatization MICONN-T-pieces as flow-reaction chamber cassette system and the use of cassettes in HPLC is descri- Separation cassette principle detail elsewhere [6,7n.

Photometric detectors - especially those with variable wavelength - are the most frequently used in HPLC. They have proven

their value in a large number of applications. In some cases, however, their performance is inadequate, for example when the absorbance of the component of interest is too small or when

traces are to be detected.

In such cases it is possible under certain circumstances to use

other detection principles, e.g. refractometry or electrochemistry.

Another possibility, which utilizes the advantages of photometric

detection, is to perform post-column derivatization in order to produce compounds which are more easily detectable. Fundamental studies on this technique have been carried out by, e.g. Frei et al [1-3], Snyder [4] and Zech and Volter [5]. The column outlet T-piece of the HPLC cassette system (Figs. 1 and 2) provides a simple method of adding a reagent solution.

256 VOL 2, MAY 1979

The typical features of the system are the novel, problem-free

method of connecting the cassettes (“needle seal”) and the fact that the column is not fitted with the usual single inlet and single

outlet, but with a system of four combined inlet/outlets (see

Rgure 1). These features lead to easy operation, increased per- formance and a high degree of flexibility.

Figure 2 shows a possible cassette arrangement for post-column

derivatization. The sample is chromatographed in the usual way;

the reagent solution is then added via the column outlet T-piece,

e.g. by means of a second pump. Eluate and reagent are mixed in the T-piece and pass through a reaction capillary into the

detector. A chromatogram run in such a way is shown in Figure 3; it shows

gentamicin after ion-pair chromatography, derivatization with o-phthalaldehyde and fluorescence detection (conditions according to [8-10] with minor modification, see Rg. 3). Gentamicin is an aminoglycoside antibiotic which is composed of three major

and several minor components.

Journal of High Resolution Chromatography & Chromatography Communications
ANALYSIS OF PURITY OF COMMERCIAL PEPTIDES

BY HIGH RESOLUTION LIQUID CHROMATOGRAPHY

Sam A. Margolis and Pamela J. Longenfaach

Center for Analytical Chemistry

National Bureau of Standards

Washington, D. C.

I . I^ITRODUCTION

The accurate description of the mechanism of action of a chemical on a physiological system - in this case neurological - requires the use of substances of defined chemical purity. It is

essential that the test substance be free from other components

capable of perturbing the test system. The purpose of these

studies is to evaluate the state of purity of peptide hormones

in an effort to increase investigator awareness of the types and

sources of peptide and non-peptide components.

The major sources of peptide impurities that occur' in peptide

synthesis include: 1) alkylation of Trp, Tyr, or Met residues

during the removal of protective groups; 2) formation of truncated

peptides by incomplete coupling or by incomplete removal of the

protective groups on the amines; 3) duplication of an amino acid,

especially during classical synthesis procedures; 4) racemization

of amino acids (esp. His) ; 5) modification of Met, Asn, or Gin

during purification; and 6) rearrangement of peptide bonds with

Ser, Thr, or Asp [!]• T’ne major sources of non-peptide impurities

are: 1) impure reagents used during peptide synthesis and purification; 2) glassware, caps and stoppers used for packaging the
peptide; and 3) reagents used for silanization of the storage

vials

II. A?PR.\ISAL OF CURRENT METriODOLOGY FOR PEPTIDE ANALYSIS

• A good method for peptide analysis should require a short

analysis time, use small amounts of sample, employ multiple quantitative methods of detection, obtain complete sample recovery, and

make available, with minimum dilution, separated components for

further analysis of biological activity, quantitation by radioimmunoassay (RIA) , or assessment of chemical composition. The ana- lytical methods used today for examining peptide homogeneity - thin

layer chromatography and paper electrophoresis - provide a high

degree of resolution. However, amino acid sequence errors that

occur during peptide manufacture may not always be detected by

these methods. Neither may errors in the amino acid sequence of

synthetic peptides be detected by amino acid analysis. Furthermore

the amino acid values reported in the literature often vary as much

as 7 percent from the theoretical value. If a given peptide con- tains 2 moles of an amino acid, a seven percent error in analysis

can translate into as much as fourteen percent of the peptide repre

senting either a deletion or an addition error. Finally, very few

of the analytical methods that are routinely used in peptide analysis detect non-peptide impurities. With the exception of the measurement of specific rotation and elemental analysis, essentially

all the methods utilize detection techniques which are specific for

amino acids or the peptide bond. Specific rotation measurements

will detect only optically active impurities, and elemental analyses are often not reported. Only high resolution liquid chromatography uses the more general quantitative detection methods , such

as ultraviolet absorption at nominal wavelengths of 215, 254, and

280 nm. Use of variable wavelength detectors can be used to give

more versatility. Other more specific quantitative techniques,

such as fluorescence measurement and post-column derivatization
reactions can be used to supplement the information obtained by use

of ultraviolet detectors.

High resolution liquid chromatography (LC) can fulfill most of

the requirements for a good analytical method for peptide analysis.

Less than thirty minutes is usually required for the analysis of

a 10-100 ug sample of a peptide. Tne components can be monitored

and quantitated at specific wavelengths (usually 213, 254 or 280

nm) , or by post-column derivatization techniques [2] . The entiresample can be recovered [3]

,

hydrolyzed, and analyzed for amino

acid composition by the method of Benson and Hare [4]

;

assessed for

biological activity; or quantitated by RIA [5]

.

The utility of high resolution LC for analysis of peptide

hormones was demonstrated by Rivier [5] for luteinizing hormone

releasing hormone (LRH) , somatostatin and thyrotropin releasing

hormone (TRH) , and by Molnar and Horvath [6] for angiotensins and

melanocyte stimulating hormone (MSH) . Other investigators have

confirmed these results and have also examined other synthetic peptides [7-12]. Krumen and Frei [11] separated analogs of oxytocin

and vasopressin [2]. Feldman [15,14] also chromatographed some of

these hormones and demonstrated that more than one chromatographic

peak can be found in commercially prepared peptides. The resolution of diastereomers of peptides was accomplished using an octadecylsilane column, with acetonitrile: ammonitim acetate buffer as the mobile phase [15]. The [D-Trp ]- and [D-Cys"^ ]- diastereomers

of somatostatin were completely separated from somatostatin and

14

the [D-Met ] -bombesin from bombesin. Most recently Takai et al.

5 5 4

[1] separated [Val ]-, [lie ]- and [D-Tyr ] -angiotensins II and

demonstrated that the tryptic peptides of [D-His]-, [D-Tyr]- and

[3 Asp] -angiotensins II could be resolved from the tryptic hydrolysates of angiotensin II. On the basis of these studies, they

demonstrated that their preparation of synthetic angiotensin II was free of diastereomers and the 3-a.spartyl derivative.

Almost every sample of commercial peptide that has been examined contained multiple impurity peaks which absorb light at 254 nm.
However, the origin, identity and structure of these conponents

have not been elucidated. Only Rivier determined the amino acid

composition of some of the components, that were separated by the

chromatography process, and measured the recovery of peptide after

chromatography [3]

Our objective was the assessment of the purity of commercial

peptides under conditions, where essentially all of the peptide was recovered, by use of a method that fulfills the requirements that

have been outlined for a good analytical procedure. This was achieved by modifying the solvent system of the high resolution LC

method of Rivier [5]Six of the nine biologically active peptides

that we have examined were obtained from more than one source.

Ever^/ peptide sample contained impurities. Some of these were

errors in peptide synthesis, and some were non-peptide components

which were introduced during purification or eluted from the

stopper.

III. METHODS AND MATERIALS

Peptides were obtained from Beckman Inst. Inc., Boehringer

Mannheim Biochemicals, Peninsula Laboratories Inc., CalbiochemBehring Corp., and Pierce Chemical Co. Tne peptides were chromatographed on an octadecylsilane column using commercial instrumentation and they were detected with a fixed wavelength detector set at

254 nm. All solvents were degassed and filtered. The solvent

system was composed of 0.1 mol/L aqueous triethylamine phosphate

(TEAP) , pH 5.3, and acetonitrile. Chromatography was done isocratically at room temperature, with acetonitrile representing 17.5,

19, or 25 percent by volume of the total solvent. Small changes in

pH resulted in significant changes in resolution and retention

times of some peptides. Fractions of the column effluent that con- tained material absorbing at 254 nm were collected, concentrated

under nitrogen, hydrolyzed, and analyzed for amino acid composition

by the method of Benson and Hare [4]

Recoveries were based on the
quantity of amino acid measured by this technique. Angiotensin I fractions were analyzed by RIA by Dr. Jean Sealey (Cornell

University)

.

IV. .APPLICATION OF HIGH RESOLUTION LIQUID CHROMATOGRAPHY TO

THE PROBLEM OF PEPTIDE HORMONE PURITY

A. Properties of the Chromatographic Method

The chromatographic technique requires small samples of 25-

100 ug and requires less than 30 minutes for the analysis of either

angiotensin I, angiotensin II, or [Val^] -angiotensin II (Figures 1 and 2) . Tne detector is sensitive to peptides which contain Tyr,

Phe, or Trp, as well as non-peptide compounds which absorb light at

254 nm. Greater than 95 percent of the angiotensin I or II applied

to the column was recovered when the main peaks were collected and

analyzed for amino acid composition. The identities of all the

chromatographic peaks that contained peptide were established in

this manner. The response of the detector at 254 nm to angiotensin

I was linear for samples of 25 to 100 ug (Figure 5) , indicating

that the method is quantitative over this range. Furthermore,

radioimmunoassay of the material in the angiotensin I peak at 20

minutes (Fig. 2) indicated that more than 95 percent of the RI.A detectable peptide was located in this peak. Approximately 0.3

percent of the total RIA activity was located in the peaks at 12,

13, and 15 minutes.

The resolution of the chromatographic system is illustrated in

Figures 1 and 2 where angiotensins I and II and [Val^] -angiotensin

II are completely resolved from each other. The baseline resolu5 5 tion of [Ile^]- from [Val ] -angiotensin II, which differ by a single methylene group, provides an example of the degree of resolution achieved by this method. Of equal importance is the baseline resolution of des [ I le^] -angiotensin II from angiotensin II

where the former (retention time 3 minutes) is present at one
r.ventieth the concentration of the angiotensin II (retention time

6.5 minutes) (Fig. 1). No amino acids or peptides were detected in

the other peaks.

B. Types and Sources of the Impurities found with Angiotensin I

Angiotensin I was extensively studied by chromatography using

several solvents in an effort to delineate the sources of impurities. Fresh solutions of angiotensin I were found to contain a single peptide component with the expected amino acid composition

(Fig. 2) when chromatographed in TEAP-acetonitrile (31:19 v/v) . .A second peptide component (retention time 25 minutes) exhibiting

pressor activity in rats and having the same amino acid composition

as angiotensin I was detected after storage of the angiotensin I as the lyophilized product at 4 °C (Fig. 2) . Although the basis for

the formation of this peptide component is not understood, it

appears that as the lyophilized angiotensin I ages it is partially

converted to this new form. The magnitude of the chromatographic

peak for this second component relative to that of the angiotensin

I peak is the same whether the lyophilized sample is dissolved

either in water or in the chromatographic solvent prior to the

chromatographic analysis. Furthermore, the formation of this com- ponent seems to be independent of the source of the angiotensin I (Table I) . The other peaks in the angiotensin I chromatogram

represent predominantly non-peptide impurities. Peptide was not

detected in these peaks by amino acid analysis. However, a small

amount of material measurable by RIA (approx. 0.5%) was detected in

the three peaks which have retention times between 12 and 15

minutes (Fig. 2). The minimum amount of peptide required for this

RIA measurement (100 pg) is not detectable by our amino acid analysis method and that amount of peptide would not account for the

magnitude of the absorbance of these components. These peaks

between 12 and 15 minutes are absent or very low in the crude

angiotensin I peptide obtained after HF cleavage of the peptide
from its solid phase support (Fig. 4) . Their intensities differ

markedly between lots from the same company and vary somewhat

between samples of the same lot (Table I) . The angiotensin I from

source 5 contained only traces of these impurities. Thus, it

appears that non-peptide impurities are introduced during the purification steps which may co-elute with trace amounts of material

measurable by RIA.

Chromatography of angiotensin I in TEAP: acetonitrile (73:25

v/v) resolved a second set of four impurities (retention times, 5,

7.5, 18, and 19,5 minutes) which elute after angiotensin I (Fig.

5) . These impurities were found to be contaminants from the serum

stopper and were present in trace amounts in the lyophilized material. ^Vhen the angiotension I was dissolved in water that was

prepared specially free of organic impurities [16] and the solution

was not permited to come in contact with the serum stopper, only

trace amounts of these impurites were detected. Subsequent exposure

of the angiotensin I solution to the stopper increased the height

of these peaks two- to four-fold (Fig. 3, Trace B) . IVhen the

stopper was transferred to a new vial and exposed to the chromatography solvent, additional amounts of these impurities were

eluted from the stopper, (Fig. 3, Trace C) . Other peptides such as somatostatin showed similar contamination with impurities originating from the serum stopper (Fig. 6)

.

C. Purity of Other Commercial Peptides

A series of peptides which are important to the neurophysiologist were also evaluated by these methods. Somatostatin and

substance P were chromatographed in TEAP -.acetonitrile (75:25 v/v).

Somatostatin contained between 8 and 10 impurities. One impurity

(retention time 16 minutes) was the deletion error peptide des-[Lys

Phe Tnr]-somatostain (Fig. 6). The other peaks contained no peptide and four of these peaks were due to the same impurities of

stopper origin as those found in angiotensin I.
Substance P contained no other peptides. However, the peak

containing the peptide appears to be associated with a partially

resolved component (Fig, 7} and must be studied further. More than

eight non-peptide peaks were also evident.

LRH is one of the purer peptides evaluated with respect to the

number of additional peaks. However, 5-6 small non-peptide peaks

were detected in addition to one major impurity peak (retention

time 26 minutes) that accounted for seven percent of the peptide

material, as estimated by amino acid analysis. This pentide impurity contained extra Gly, one extra Glu and one extra Ser in

addition to the expected amino acid composition of LRH (Fig, 8).

A second lot of LRH also contained a peptide impurity - des-[Gly]-

LRH- which represented nine percent of the total peptide as estimated by amino acid analysis.

The eledoisin sample contained only a single peptide peak which

had the proper amino acid composition. However, it contained eight

or more components that were devoid of amino acid content (Fig. 9)

[Leu]- and [Met] -enkephalin samples appeared to be the purest

of the peptides which we examined. Each sample contained a single

peptide peak and five to six very small peaks which did not contain

peptide or amino acids (Fig. 10) . The peptides obtained from

source 1 appeared purer than those obtained from sources 2 and 3.

V. DISCUSSION AND CONCLUSIONS

The use of the TEAR- acetonitrile solvent system with an octadecylsilane column confirms and extends the observations of Rivier

[3]

Peptides collected after chromatography in this buffer can be

analyzed and quantitated directly by RIA, and preliminary results

with angiotensin I and II indicate that with pretreatment (solvent

removal) these samples can also be analyzed by bioassay procedures

for biological activity. The peptide fractions can be dried under

nitrogen, hydrolyzed directly in 6 mol/L HCl and analyzed for amino

acid composition. More than 95 percent of the peptide is recovered.
The magnitude of the ultraviolet absorbance is proportional to the

concentration in the range of 25 to 100 yg of peptide and the

analysis time is less than 60 minutes.

The resolution of this method is demonstrated by the separaS 5 tion of [Val ]- and [lie ] -angiotensin II and by the resolution of

such impurities as des- [I le^] -angiotensin I, des- [Gly] -LRH, LRH

with four extra amino acids, des-[Lys, Phe, Thr] -somatostatin, des-

[Ser] -somatostatin and the several non-peptide impurities that are

present in each of these hormone preparations which, according to

the suppliers, were pure and homogenous. The lack of peptide

purity or the presence of a number of chromatographically distinct

components confirms the results of other investigators who examined

commercial material [3,9-14]. However, these investigators did not

examine the chemical composition of these peaks.

The component in angiotensin I which has a retention time of

25 minutes and appears to form upon storage of the lyophilized

material, does not seem to be a chromatographic artifact since the

other peptides that we analyzed did not exhibit similar behavior

(Fig. 6-10). This second component of angiotensin I may be 3-[Asp]

angiotensin I. It has the same amino acid analysis as angiotensin

I and its position in the chromatogram relative to angiotensin I is

similar to that of the S-[Asp]- derivative of [Asp-Arg-Val-Tyr]

relative to its a derivative [1]

.

Several of the non-peptide impurities in angiotensin I appeared to have been introduced during the purification of the pep

tide. This was indicated by their absence in crude angiotensin I.

Furthermore, e'ach lot of peptide contained a different proportion

of each impurity peak. The variation in the amount of the nonpeptide components is illustrated in Table I for the impurity peaks

at 12, 13, and 15 minutes. Both inter- lot and inter-vial variations were observed in the peak areas and one lot contained negligible amounts of these peaks, ’'/hen the cornponents in the angiotensin I impurity peaks eluting at 12, 13, and 15 minutes were

concentrated under nitrogen and chromatographed, they were not
detected. Thus, these peaks probably represent volatile impurities

that were introduced during the peptide purification process and

were incompletely removed to various degrees by lyophilitation

Another set of non-peptide impurities found in several different peptide preparations was eluted from the stopper. The small

amounts of these stopper impurities, which were present in the

lycphilited mass of peptide, may have been introduced during the

filling or lyophilitation process, or may have been present in the

final peptide solution and adsorbed onto the stopper during the

lyophilitation process. Their presence is not restricted to angiotensin I. They have been observed also in somatostatin and other

peptide preparations, particularly after the peptide solution was permitted to come in contact with the serum stopper.

Because of the volatility of the peaks at 12, 15, and IS minutes that were obser'/ed in angiotensin I samples, we have been

unable to establish their identity. However, these components

exhibited ver>' small but significant amounts of material measurable

by the radioimmunoassay for angiotensin I; suggesting the presence

of trace amounts of peptide. However, no peptide was detected in

these peaks by amino acid analysis. Therefore, the absorbance of

these peaks was due to something other than peptide, leading to the

conclusion that these three peaks represent trace peptide impurities

which CO -chromatograph with non-peptide components.

We have attempted to identify some of these impurities by use

of mass spectrometr>'. Preliminary evidence for angiotensin I indicates that the lyophiliied mass of peptide contains, in addition to

the peptide, dimethyls! loxane polymers, acetic acid, tri ethyl amine,

and material which decomposes above 300 ®C releasing benzyl alcohol.
ACKNOWLEDGEMENT

We thank Dr. Jean Sealey for kindly performing the radioimmunoassays, Dr. M. Khosla for kindly performing the bioassays on angiotensin I, and Dr. E. rVhite for the mass spectrometry. We also

thank Dr. Jeffery Barker for his encouragement and for generous

supplies of peptide hormones. This research was supported by a research agreement with the National Heart, Lung and Blood Institute, NIH USPHS contract l-Y-Ol-HV-70042-00 . Identification of

any commercial product does not imply recommendation or endorsement

by the National Bureau of Standards nor does it imply that the

material or equipment identified is necessarily the best available

for the purpose

.

REFERENCES

1. M. Takai, S. Kumagaye, T. Kishida, and S. Sakakibara, in Peptide Chemistry 1973, Proceedings of the 16th Symposium on Peptide Chemistry (N, Itumiya, ed.) Protein Research Foundation, Minoh, Osaka, 1979, p. 67.

2. R. W. Frei, L. Michel, and W. Santi, J. Chromatogr ., 126 : 665

(1976}

.

3. J. E.'Rivier, J. Liq. Chromatogr ., 345 (1978).

4. J. R. Benson, and P. E. Hare, Proc. Natl. Acad. Sci . , U. S

.

, 619 (1975].

5. R. Burgus and J. E. Rivier, ' Teptides 1976* ' (.A. Loffett, ed.]

Editions d 1 'university de Bruxelles, Belgium, p. 85.

6. I. Molnar, and C. Horvath, J. Chromatogr ., 142 : 623 (1977].

7. L. R. Pickart and M. M. Tnaler, Prep. Biochem ., 397 (1975].

8. K. Tsuji and J. H. Robertson, J. Chromatogr ., 112 : 663 (1975].

9. W. S. Hancock, C. A. Bishop, and M. T. W. Hearn, Febs . Lett .

,

7^; 139 (1976]

.

10.

J. J. Hansen, T. Greibokk, B. I. Currie, K. N. Johansson, and

K. Folkers, J. Chromatogr., 155 : 155 (1977].
- 1 . N . Krumen and R. W. Frei, J. Ccromatogr., 132: 27 (19"7

W . S. .Hancock, C. A. Bishop, L. J. Meyer, D. R. K. Hard

and M. T. W. Hearn, J. Chromatogr., 161: 291 CIS^S)

13

TJ . Feldman, M. Cohn, and D. Blair, J. Lio . Chromatogr.,

:i9 Sj .

1-. J. reldman, and M. L. Cohn, in 3iological/3iomedical Anplications of Liquid Chromatography (G. L. Hawk, ed.), Marcel

Dekker Inc., New York, 1979, p. 225.

15. J. Rivier and R. Burgus, in Sio logical/ Biomedical Applications

of Liquid ‘Chromatography [G. L. Hawk, ed.), Marcel Dekker Inc.,

New York, 1979, p. 147.

16. 5. N. Chesler, 3. H. Gump, H. S. Hertc, W. E. May,

S. M. Dyscel, D. P. Enagonio, Trace Hydrocarbon Analysis: The

National Bureau of Standards, Prince William Sound/Northeastem Gulf of Alaska Baseline Study , N3S Technical Note 889 , U. S. Gov't. Printing Office, Washington, D.C., 1976.
TABLE I Magnitude of Impurity Peaks in Angiotensin I

Relative Peak Area (Al'’ = 1000)

Actual

Peak Area

Source Lot 12 min. 13 min. 15 min. 25 min. Mi

1 A 20 159 61 133 540

17 247 81 116 580

20 208 65 105 611

B 2 52 19 121 630

2 A 2 69 19 118 566

4 130 37 103 563

9 100 27 116 558

B 5 74 23 92 739

5 69

% 21 116 773

C 6 74 16 102 724

7 64 16 103 685

3 A <1 9 <1 120 548

<1 <1 <1 128 550

^ach value represents the average of similar values of two separate determinations on the same sample. Every sample was dissolved

in water free of trace organic compounds. The concentration of

the peptide was 0.5 mg/mL based on the peptide content assigned by

the supplier. A 50 uL aliquot of each solution was chromatographed for each analytical determination.

AI = angiotensin I (ret. time = 20 min.)
FIGURE CAPTIONS

FIG. I. Resolution of [Ile^]- and [Val^] -angiotensin II. Twentyfive ug of peptide, dissolved in the chromatographic solvent, was applied to an octadecylsilane column that was equilibrated with

acetonitrile :TE.-\P (19:81 v/v) at a flow rate of 1 mL/min. Detection was at 0.02 absorbance units full scale (AUFS) . The peptide

peaks were identified by amino acid analysis.

FIG. 2. The Effect of Storage at 4 on Commercial Lyophilized

Angiotensin 1. Twenty-five yg of peptide, dissolved in the

chromatographic solvent, was analyzed on an octadecylsilane column

that was equilibrated with acetonitrile: TEAP (19:81 v/v) at a flow

rate of 1 mL/min. Detection was at 0.02 AUFS. Left trace:

angiotensin I at the time of receipt. Right trace: angiotensin 1 after eight weeks of storage at 4 °C.

FIG. 3. Load - Response Curve for Angiotensin I. Incremental

quantities of angiotensin I were chromatographed as described in

Figure 1.

FIG. 4. Chromatogram of Crude Angiotensin I. Twenty-five yg of

lyophilized peptide, obtained by HF cleavage from the solid phase,

was dissolved in the chromatographic solvent and applied to an octadecylsilane column that was equilibrated with acetonitrile ;TE.A.P (19:^81 v/v) at a flow rate of 1 mL/min. Detection was at 0.02

AUFS*.

FIG. 5. Identification of the Angiotensin I Impurity Peaks iNTiich are Eluted From the Serum Stopper. Fifty microliters of each sam- ple was chromatographed on an octadecylsilane column equilibrated

with acetonitrile : TEAP (25:75 v/v) at a flow rate of 1.7 mL/min.

Detection was at 0.02 AUFS. Trace A : Angiotensin I dissolved in

organic compound free water at a concentration of 0.5 mg/mL without

contact with the serum stopper. Trace B : Angiotensin I solution

part of which was used in Trace A, after contact with the serum

stopper. Trace C : Chromatographic solvent after contact with the

stopper that was used in the preparation of the sample for Trace B.

FIG. 6. Resolution of Impurity Peaks in Commercial Somatostatin.

Twenty-five yg of somatostatin (SS) , dissolved in the chromatographic solvent, was applied to an octadecylsilane column that was

equilibrated with acetonitrile : TEAP (25:75 v/v) at a flow rate of

1 mL/min. Detection at 0.05 .4UFS. The peptide peaks were identified by amino acid analysis.
FIG. 7. Resolution of Impurity Peaks in Commercial Substance P.

Twenty- five ug of substance P (SP) , dissolved in the chromatographic solvent, was applied to an octadecylsilane column that was

equilibrated with acetonitrile : TEAP (25:73 v/v) at a flow rate of

1 mL/min. Detection was at 0.01 AUFS. The peptide peaks were

identified by amino acid analysis.

FIG. 8. Resolution of Impurity Peaks in Commercial LRH. Twentyfive ug of LRH, dissolved in the chromatographic solvent, was

applied to an octadecylsilane column that was equilibrated with

acetonitrile: TEAP (17.5:82.5 v/v) at a flow rate of 1 mL/min.

Detection was at 0.05 .AUFS. The peptide peaks were identified by

amino acid analysis.

FIG. 9. Resolution of Impurity Peaks in Eledoisin. Twenty-five

U'g of Eledoisin (E) , dissolved in the chromatographic solvent, was

applied to an octadecylsilane column that was equilibrated with

acetonitrile: TEAP (19:81 v/v) at a flow rate of 1 mL/min. Detection was at 0.02 .AUFS. The peptide peak was identified by amino

acid analysis.

FIG. 10. Resolution of Impurity Peaks in Different Commercial

Preparations of [Met]- and [Leu] -Enkaphalins . Twenty-five ug of

each peptide, dissolved in the chromatographic solvent, was applied

to an octadecylsilane column that was equilibrated with acetonitrile: TEAP (17.5:82.5 v/v) at a flow rate of 1 mL/min, Detection

was at 0.02 AUFS. The peptide peak was identified by amino acid

analysis

.
Mixture 1 :1

[Iles]-An

[Val®]-A2
Time

(min)

Time

(min)
300

200

100

0 25 50 75

Load (ug)

100
Time (min)

Time

(min)
LO

CM

<

Time

(min)
Time

(min)
i

Time

(min)
Time

(min)

Time

(min)

'j.i. TePT. 0- COMM.

j 1. P'JBLiCATtON OR REPORT NO. | ------ --i BIBLIOGRAPHIC DATA J j SHEET j N.B.S. IR-79-1947 ;

4. TITLE AND SUBTITLE

Final .Report on Agreement //YOl-HV-70042

Development of a Standard Reference Material for

Angiotensin I

5. Puuiication Data

5. P;no." ,ir)5 Oiganuatioo Cc ci

7. AUTHORiS)

Sam A. Margolis and Robert Schaffer

8. Perrorming Organ. Repor: No.

3. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASKiNGTON, DC 2023X

10, P'o; set, Taaw,."Work Unit No.

11. Contraci/Grant No.

12. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS rSfree?, City. State. ZIP) 13. Type of Report & Period Covered

14. Sponsoring Agency Ccce

•

15. SUPPLEMENTARY NOTES

rj Dccutnent describes a computer program: SF-185, FIPS Software Summary, is attached.

16. ABSTRACT (A 200-w’ord or leas taccuai summary of most signiticant miormation» If docume.it includes a significant bibliography jt literature survey, mention it here.) The primary problem in the preparation of an Angiotensin I standard appears

to be the spontaneous a to B rearrangement of the amide linkage of the carboxyl

group of the aspartic acid residue. This rearrangement results in an enhanced

biological activity of the Angiotensin I preparation in proportion to the content

of the rearranged product. Since it is 50 percent more active than Angiotensin I,

if present as 30 percent of the preparation, then samples would show 15 percent

higher activity in the bioassay. However, RIA does not distinguish between the

two forms (personal communication from Dr. J. Sealey); hence, activity by RIA

would be unchanged regardless of the ratio of these isomers. The impurities

present in the Angiotensin I do not appear to modify the sensitivity of the RIA

assay for Angiotensin I. Thus if the peptide content is accurately stated then

the peptides we have analyzed appear to be suitable for use as RIA standards,

but not as standards for biological assay unless their content of [3-Aspj - Angiotensin I is periodically assessed. With this stipulation. Angiotensin I now available can be certified as a standard.

17. KEY WORDS (Six ro nreire enrr;>»; ziphabeiica! order; capitalize only the (irat letter of the first key vord unices a proper name;

separated by semicolons) /\n g

1 Q t OH s i 0 s ; c T i t or i 3 f0 r SRM; HPLC; peptide purity.

18. AVAILABILITY rxr Unlimited IS. SECURITY CLASS

(THIS REPORT)

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