Drug Testing

Short Communication and Analysis

Received: 27 June 2010 Revised: 15 October 2010 Accepted: 15 October 2010 Published online in Wiley Online Library: 10 December 2010

(www.drugtestinganalysis.com) DOI 10.1002/dta.233

Identification of CJC-1295, a
growth-hormone-releasing peptide,
in an unknown pharmaceutical preparation
John Henninge,a∗ Milaim Pepaj,b Ingunn Hullsteina
and Peter Hemmersbacha,c
Several peptide drugs are being manufactured illicitly, and in some cases they are being made available to the public before
entering or completing clinical trials. At the request of Norwegian police and customs authorities, unknown pharmaceutical
preparations suspected to contain peptide drugs are regularly subjected to analysis. In 2009, an unknown pharmaceutical
preparation was submitted for analysis by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS).
The preparation was found to contain a 29 amino acid peptide with a C-terminal amide function. Based on the interpretation
of mass spectrometric data, an amino acid sequence was proposed. The sequence is consistent with a peptide currently
marketed under the name CJC-1295. CJC-1295 is a releasing factor for growth hormone and is therefore considered a Prohibited
Substance under Section S2 of the WADA Prohibited List. This substance has potential performance-enhancing effects, it is
readily available, and there is reason to believe that it is being used within the bodybuilding community. Copyright

c 2010
John Wiley & Sons, Ltd.

Keywords: CJC-1295; growth hormone; doping control; Orbitrap; LC-MS/MS

Introduction consisted of approximately 2 mg of a white, flocculent powder

Recent advances in genomics and proteomics have led to an
increased focus on the development of peptide therapeutics. Due
to improved and simplified manufacturing techniques, disregard
for patent protection, and a growing global market for non-
approved drugs, several peptide drugs are being manufactured
illicitly and are being made available to the public before entering
or completing clinical trials. Some of these drugs may be misused
to enhance sports performance, and thus they are prohibited by
the World Anti-Doping Agency (WADA).[1]
Norwegian police and customs authorities are striving to curtail
the trafficking and distribution of performance-enhancing drugs.
At their request, unknown pharmaceutical preparations suspected
to contain peptide drugs are regularly subjected to analysis. Over
the past five years, the majority of such preparations submitted for
analysis have been shown to contain human growth hormone
(hGH) and the skin tanning drug Melanotan-II, respectively.
Preparations containing human chorionic gonadotrophin (hCG),
insulin-like growth factor-1 (IGF-1) and the growth hormone
releasing peptides GHRP-2[2] and GHRP-6[3] have also been
encountered by the WADA-accredited laboratories.
contained in an unmarked glass vial, sealed by an aluminium
cap with an orange plastic top. The vial is shown in Figure 1.
An aqueous solution of the contents was prepared at a nominal
concentration of 10 µg/mL. A 50 µl aliquot of the solution was
diluted 1 : 9 with 50 mM aqueous ammonium bicarbonate and
subjected to enzymatic digestion. Sequencing grade modified
trypsin (Promega, Madison, WI, USA) was added at a 1 : 20 ratio
(enzyme to protein), and the sample was incubated for 4 h at 37 ◦ C.
The solution of the unknown substance and the tryptic
digest were both subjected to analysis on a Dionex Ultimate
3000 liquid chromatography system (Dionex, Sunnyvale, CA,
USA) coupled to a Thermo LTQ Orbitrap XL mass spectrometer
(ThermoFisher Scientific, Bremen, Germany). The instrument
system was calibrated using the manufacturer’s calibration
mixture, and the mass accuracy was determined to be <5 ppm
during the period of analysis. A sample volume of 5 µl was injected
onto the system. The chromatographic separation was performed
on an LC Packings PepMap 100 C18 column (150 × 1 mm, 3 µm
particle size) (Dionex, Sunnyvale, CA, USA) with mobile phases
consisting of 0.1% formic acid in water (A) and 0.1% formic acid
in acetonitrile (B) at a total flow rate of 40 µl/min. The elution

Experimental
All reagents used were of analytical grade and all solvents were
of HPLC grade. Water was obtained using a Millipore purification
system (Millipore, Bedford, MA, USA).
An unknown pharmaceutical preparation was submitted for
analysis by liquid chromatography-high resolution tandem mass
∗
Correspondence to: John Henninge, Norwegian Doping Control Laboratory,
Oslo University Hospital, Aker, Postboks 4959 Nydalen, N-0424 Oslo, Norway.
E-mail: john.henninge@h-lab.no
a Norwegian Doping Control Laboratory, Oslo University Hospital
b Hormone Laboratory, Oslo University Hospital
647

spectrometry (LC-HRMS/MS). Upon reception, the preparation c School of Pharmacy, University of Oslo

Drug Test. Analysis 2010, 2, 647–650 Copyright

c 2010 John Wiley & Sons, Ltd.
 Drug Testing
and Analysis J. Henninge et al.

at m/z = 842.4825, which corresponds to a monoisotopic

Figure 1. Vials found to contain hGH (left), CJC-1295 (centre) and
Melanotan-II (right).
employed a linear gradient starting at 5% B, increasing to 50%
B in 20 min. The mass spectrometer was operated in positive
electrospray ionisation (ESI+) mode, with a spray voltage of 4 kV
and a capillary temperature of 200 ◦ C. Nitrogen was used as sheath
gas at a flow setting of 8 arbitrary units. For data acquisition, a data-
dependent MS/MS scan setting was applied. High resolution mass
spectra were acquired in profile mode (scan range m/z 300–2000)
in the Orbitrap analyzer with the resolution set at 30 000, followed
by product ion scans of the two most abundant precursor ions in
the linear ion trap, using a normalized collision energy of 35% and
an isolation width of 3 Da.
Results and Discussion
The high resolution full-scan mass spectrum of the intact peptide,
shown in Figure 2, indicates a quadruply charged ion [M+4H]4+
molecular mass of 3365.8989 Da. The experimentally determined
monoisotopic mass is consistent with the elemental composition
C152 H252 N44 O42 (calculated mass 3365.8936 Da, error 1.6 ppm).
Tryptic digestion yielded three peptides which were separated
by liquid chromatography. The tryptic peptides were named
peptide A, B, and C. The base peaks of their mass spectra were
doubly charged ions [M+2H]2+ at m/z = 667.8217 (peptide A),
m/z = 493.3105 (peptide B) and m/z = 543.3355 (peptide C),
respectively. The observed ion at m/z = 667.8217 of peptide A
corresponds to a monoisotopic mass of 1333.6277 Da, which
is consistent with the elemental composition C61 H87 N15 O19
(calculated mass 1333.6303 Da, error 1.9 ppm). The product ion
spectrum of m/z = 667.82 is shown in Figure 3a.
The doubly charged ion at m/z = 493.3105 of peptide B
corresponds to a monoisotopic mass of 984.6054 Da, which
is consistent with the elemental composition C43 H80 N14 O12
(calculated mass 984.6080 Da, error 2.6 ppm).The product ion
spectrum of m/z = 493.31 is shown in Figure 3b.
The doubly charged ion at m/z = 543.3355 of peptide C
corresponds to a monoisotopic mass of 1084.6553 Da, which
is consistent with the elemental composition C48 H88 N14 O14
(calculated mass 1084.6605 Da, error 4.8 ppm).The product ion
spectrum of m/z = 543.34 is shown in Figure 3c.
For each of the three tryptic peptides, a proposed amino acid
sequence and a rationale for the observed fragmentation pattern
is shown in Figure 4.
A discrepancy of one mass unit is observed between the mass of
the intact peptide and the sum of masses of the tryptic peptides,
corrected for the water added during cleavage of two peptide
bonds. Comparison of the elemental compositions reveals that
one nitrogen atom in the intact peptide is substituted by oxygen in

CJC_intakt #654-692 RT:14.88-15.34 AV:15 NL:3.32E7 CJC_intakt #654-692 RT:14.88-15.34 AV:15 NL:3.32E7
T:FTMS + p ESI Full ms [300.00-2000.00] T:FTMS + p ESI Full ms [300.00-2000.00]
842.7317 842.7317
z=4 z=4

32000000 30000000

30000000 25000000
843.2294
z=4
28000000 20000000 842.4825
z=4
Intensity

26000000 15000000
843.4797
z=4
24000000
10000000

22000000 843.7305
z=4
5000000

20000000
0
838 839 840 841 842 843 844 845 846 847 848
Intensity

18000000
m/z

16000000

14000000 674.3867 1123.3052

z=5 z=3
12000000

10000000

8000000

6000000

4000000

2000000

0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
648

Figure 2. High resolution full-scan mass spectrum of the intact peptide found in the unknown pharmaceutical preparation.

www.drugtestinganalysis.com Copyright

c 2010 John Wiley & Sons, Ltd. Drug Test. Analysis 2010, 2, 647–650
 Drug Testing
Identification of CJC-1295 in an unknown pharmaceutical preparation and Analysis

CJC_digest #725 RT:14.8 AV:1 NL:1.99E5

F: ITMS + c ESI d Full ms2 667.82@cid35.00 [170.00-1350.00] 801.4
(a)
180000
160000
140000
120000
Intensity

100000
80000
802.3
60000 914.5
534.2 654.3
40000
350.1 659.0 915.4
506.2 985.5 1100.6
20000 393.1
421.1 489.2 553.2 1101.5
207.2 235.2 258.2 303.2 338.3 592.1 653.6 681.3 727.9 764.4 783.4 803.0 875.3 892.3 944.9 979.3 997.4 1040.5 1082.5 1171.9 1204.5 1247.6
0
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350
m/z
CJC_digest #590 RT:13.20 AV:1NL:6.33E4
F: ITMS + c ESI d Full ms2 493.31@cid35.00 [125.00-1000.00] 758.3

(b) 60000
55000
50000
45000
857.5
40000
Intensity

35000 484.7
30000
25000 759.5
645.3
20000
858.5
15000
574.3
10000 446.2 646.4
333.3 485.4
5000 183.3 228.2
246.2 341.3 394.5
412.3 476.1 575.4 653.4 760.4 859.6
155.3 296.5 313.3 486.0 523.3 556.4 623.1 673.5 722.5 740.4 811.5 840.7 885.6
0
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
m/z
CJC_digest #695 RT:14.41 AV:1 NL:1.12E5
F: ITMS + c ESI d Full ms2 543.34@cid35.00 [135.00-1100.00]
844.5
(c) 110000
100000
957.5
90000 534.8

80000
70000
Intensity

488.3
60000
731.4
50000 845.4
40000 603.4
958.5
30000 598.3
535.4
20000 489.4 732.4
375.2 604.4 714.4
337.4 581.3
10000 242.2 262.2 466.5 824.5
175.3 197.2 320.3 376.4 421.4 525.7
563.3
711.5 809.2 846.6
911.4
940.7 967.4
263.4 605.8 653.1 733.6 986.4
0
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100
m/z

Figure 3. Product ion spectra of m/z = 667.82 (a), m/z = 493.31 (b) and m/z = 543.34 (c); the doubly charged ions [M+2H]2+ of peptides A, B and C,
respectively.

the tryptic digest. This indicates that a C-terminal amide function is CJC-1295 was developed by ConjuChem Biotechnology Inc.
present in the intact peptide, but not in the digest. The mass spectra (Montreal, Canada) and in its original form, it utilizes a novel
of the tryptic peptides all display y2 and y3 fragments consistent bioconjugation technology referred to as Drug Affinity Construct
with C-terminal carboxylic acids, confirming that deamidation (DAC ). A maleimidopropionamide derivative of lysine at the C-
occurs. terminus allows it to bind covalently to albumin in vivo, thereby
From the above results, it can be determined that the unknown significantly extending its half life.[5,9] However, most products
substance is a 29 amino acid peptide with a C-terminal amide currently sold as CJC-1295, including the substance identified in
function. Interpretation of the mass spectrometric data leads to this study, are non-approved drugs lacking the DAC feature.
the proposed amino acid sequence of the intact peptide shown in CJC-1295 was withdrawn from clinical trials in 2006 and is not
Figure 5. currently approved for human use.[10]
Tryptic cleavage occurs predominantly at positions 11 and
20, as mass the spectrometric data show that peptides B and
C both contain intact N-terminal lysine residues. Although the Conclusion
experimental conditions appear to favour cleavage at arginine,
An unknown pharmaceutical preparation was subjected to analysis
peptides arising from alternate or additional cleavage at lysine
to determine whether it contained prohibited substances. The
residues were also observed at low abundance. preparation was found to contain a peptide which had not
The amino acid sequence proposed for the unknown substance previously been encountered. The experimentally determined
is consistent with a peptide currently marketed under the molecular mass and the observed fragmentation patterns are
name CJC-1295. It is a synthetic 29 amino acid analogue of consistent with the amino acid sequence of a peptide commonly
growth hormone releasing hormone (GHRH), with amino acid referred to as CJC-1295. Its identity could not be unequivocally
substitutions at positions 2, 8, 15 and 27.[4] It has been shown to confirmed, as certified reference material was not available.
increase serum levels of growth hormone.[5,6] Its mechanism of CJC-1295, whether with or without the DAC feature, is a
action is shared with GHRH and the synthetic analogues GRF1 – 29 releasing factor for growth hormone and should therefore be
and tesamorelin (TH9507), but it is distinct from the ghrelin class regarded as a Prohibited Substance under section S2 of the WADA
649

of growth hormone secretagogues.[4,7,8] Prohibited List.[1] Although there is currently no indication that

Drug Test. Analysis 2010, 2, 647–650 Copyright

c 2010 John Wiley & Sons, Ltd. www.drugtestinganalysis.com
 Drug Testing
and Analysis
Figure 4. Proposed amino acid sequences and assignment of the fragments observed in the product ion spectra of peptides A, B, and C.
Figure 5. Proposed amino acid sequence of the intact peptide.
CJC-1295 is being misused in competitive or elite sports, it has
potential performance-enhancing effects. It is readily available, as
this study clearly shows, and there is reason to believe that it is
being used within the bodybuilding community. The metabolism
and excretion of CJC-1295 should be investigated in order to
determine how its misuse best could be disclosed in sports drug
testing.
References
[1] World Anti-Doping Agency, The 2010 Prohibited List. Avail-
able at: http://www.wada-ama.org/rtecontent/document/2010
List En.pdf [27 June 2010].
[2] A. Thomas, M. Kohler, J. Mester, H. Heyer, W. Schänzer, M. Petrou,
M, Thevis, Drug Test. Anal. 2010, 2, 144.
[3] J. Henninge, I. Hullstein, G. Hagelin, P. Hemmersbach, in Recent
Advances in Doping Analysis (16), (Eds: W. Schänzer, H. Geyer,
A. Gotzmann, U. Mareck), Sportverlag Strauss: Cologne, 2008,
pp. 351–353.
650
www.drugtestinganalysis.com Copyright

c 2010 John Wiley & Sons, Ltd.
J. Henninge et al.
[4] L. Jetté, R. Léger, K. Thibaudau, C. Benquet, M. Robitaille, I. Pellerin,
V. Paradis, P. van Wyk, K. Pham, D. P. Bridon, Endocrinology 2005,
146, 3052.
[5] S. L. Teichman, A. Neale, B. Lawrence, C. Gagnon, J. P. Castaigne,
L. A. Frohman, J. Clin. Endocr. Metab. 2006, 91, 799.
[6] L. Sackman-Sala, J. Ding, L. A. Frohman, J. J. Kopchick, Growth Horm.
IGF Res. 2009, 19, 471.
[7] F. Cordido, M. L. Isidro, R. Nemiña, S. Sangiao-Alvarellos, Curr. Drug.
Disc. Technol. 2009, 6, 34.
[8] M. Jansen, I. Darby, T. Abribat, P. Dubreuil, E. S. Ferdinandi,
J. G. Hardy, Int. J. Pharm. 2004, 276, 75.
[9] M. Alba, D. Finitini, A. Sagazio, B. Lawrence, J. P. Castaigne,
L. A. Frohman, R. Salvatori, Am. J. Physiol. Endocr. Metab. 2006, 291,
E1290.
[10] ClinicalTrials.gov, A service of the US National Institutes of Health.
Available at: http://clinicaltrials.gov/ct2/show/NCT00267527 [27
June 2010].
Drug Test. Analysis 2010, 2, 647–650