Identification, characterization and quantification of process-related and degradation impurities in lisdexamfetamine dimesylate: Identifiction of two new compounds

Article abstract of DOI:10.3390/molecules23123125

Twelve impurities (process-related and degradation) in lisdexamfetamine dimesylate (LDX), a central nervous system (CNS) stimulant drug, were first separated and quantified by high-performance liquid chromatography (HPLC) and then identified by liquid chromatography mass spectrometry (LC-MS). The structures of the twelve impurities were further confirmed and characterized by IR, HRMS and NMR analyses. Based on the characterization data, two previously unknown impurities formed during the process development and forced degradation were proposed to be (2S)-2,6-di-(lysyl)-amino-N-[(1S)-1-methyl-2-phenyl ethyl]hexanamide (Imp-H) and (2S)-2,6-diamino-N-[(1S)-1-methyl-2-(2-hydroxyphenyl)ethyl] hexanamide (Imp-M). Furthermore, these two compounds are new. Probable mechanisms for the formation of the twelve impurities were discussed based on the synthesis route of LDX. Superior separation was achieved on a YMC-Pack ODS-AQ S5 120A silica column (250 × 4.6 mm × 5 μm) using a gradient of a mixture of acetonitrile and 0.1% aqueous methanesulfonic acid solution. The HPLC method was optimized in order to separate, selectively detect, and quantify all the impurities. The full identification and characterization of these impurities should prove useful for quality control in the manufacture of lisdexamfetamine dimesylate.

Full text of DOI:10.3390/molecules23123125

molecules
Article
Identification, Characterization and Quantification of
Process-Related and Degradation Impurities in
Lisdexamfetamine Dimesylate: Identifiction of Two
New Compounds
Shenghua Gao 1, Lili Meng 1, Chunjie Zhao 2, Tao Zhang 1, Pengcheng Qiu 1,* and Fuli Zhang 1,
*
1
Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry,
No. 285 Gebaini Road, Shanghai 201203, China; shh_gao@163.com (S.G.); 18221816872@163.com (L.M.);
zt0414@126.com (T.Z.)
College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China; zcjjljj@sina.com
2
*
Correspondence: pcqiu@aliyun.com (P.Q.); zhangfuli1@sinopharm.com (F.Z.);
Tel.: +86-21-2057-2000 (ext. 5077) (P.Q.); +86-21-2057-2000 (ext. 5036) (F.Z.)
Academic Editor: In-Soo Yoon
Received: 4 November 2018; Accepted: 27 November 2018; Published: 29 November 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Twelve impurities (process-related and degradation) in lisdexamfetamine dimesylate
(LDX), a central nervous system (CNS) stimulant drug, were first separated and quantified by
high-performance liquid chromatography (HPLC) and then identified by liquid chromatography
mass spectrometry (LC-MS). The structures of the twelve impurities were further confirmed
and characterized by IR, HRMS and NMR analyses. Based on the characterization data, two
previously unknown impurities formed during the process development and forced degradation were
proposed to be (2S)-2,6-di-(lysyl)-amino-N-[(1S)-1-methyl-2-phenyl ethyl]hexanamide (Imp-H) and
(2S)-2,6-diamino-N-[(1S)-1-methyl-2-(2-hydroxyphenyl)ethyl] hexanamide (Imp-M). Furthermore,
these two compounds are new. Probable mechanisms for the formation of the twelve impurities
were discussed based on the synthesis route of LDX. Superior separation was achieved on a
YMC-Pack ODS-AQ S5 120A silica column (250
×
4.6 mm
×
5 µm) using a gradient of a mixture
of acetonitrile and 0.1% aqueous methanesulfonic acid solution. The HPLC method was optimized
in order to separate, selectively detect, and quantify all the impurities. The full identification and
characterization of these impurities should prove useful for quality control in the manufacture of
lisdexamfetamine dimesylate.
Keywords: lisdexamfetamine dimesylate; impurities; structural elucidation; forced degradation;
HPLC validation
1. Introduction
Lisdexamfetamine dimesylate (LDX; formerly NRP-104), (2S)-2,6-diamino-N-[(1S)-1-methyl-
2-phenylethyl]hexanamide dimethanesulfonate) is
a novel, long-acting, central nervous
system (CNS) stimulating drug with low toxicity used as an abuse-resistant treatment of
attention-deficit/hyperactivity disorder (ADHD). LDX is a therapeutically inactive amphetamine
prodrug, and the pharmacologically active D-amphetamine is gradually released by rate-limited
hydrolysis following ingestion [1]. The drug, originally developed by Shire Development Inc. (London,
UK) and New River Pharmaceutical Inc. (Washington, DC, US) is currently marketed under the trade
name of Vyvanse since its launch in February 2007 [2,3].
The industrial manufacturing process of LDX was developed by New River Pharmaceutical Inc.
(Figure 1) [2]. Impurities in drugs are closely related to their adverse reactions and pharmacological
Molecules 2018, 23, 3125; doi:10.3390/molecules23123125
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3125
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activity. For example, degradation products, precursors, and byproducts in drugs can produce
fatal immune responses, which may be responsible for some clinical allergic reactions [ ]. After a
comprehensive literature survey, we found that only one patent cursorily referred to six impurities
of LDX [ ]. Unfortunately, there was no information about the synthesis and spectroscopic data of
LDX process-related and degradation impurities. There was only one analytical method available for
quantitative analysis of LDX in the literature [ ]. However, the paper only focused on comparison
4,5
3
6
of CAD and UV detectors, but did not include information on process-related impurities of LDX.
Furthermore, we did not get good separation resolution between LDX and process-related impurities
according to the literature. According to the guidelines recommended by the International Conference
on Harmonization (ICH), impurities present in drug substances exceeding the accepted level of 0.1%
should be identified and characterized [7]. Hence, a thorough study was conducted to develop an
effective and sensitive method for separation and identification of impurities in LDX.
Figure 1. The synthesis route of LDX. Reagents and conditions: (a) Boc2O, acetone/2N NaOH,
25 ◦C, 4 h, 94%–98%; (
b
) (i) NaBH(AcO)3, DCM, rt., 9h; (ii) THF, 36% hydrochloric acid, 73%–78%;
◦
(c) ammonium formate, MeOH, 65 C, 3 h, 92%–96%; (
d
) (i) EDCI, HOBt, NMM, DMF, rt., 20 h, 92–95%;
◦
(ii) recrystallization (acetone:n-heptane = 1:10, v/v); (e) MeSO3H, THF, 50 C, 6 h, 95%–96%.
This study aimed to: (1) identify impurities formed during the preparation of LDX and its
forced degradation study; (2) characterize and confirm structures of process-related and degradation
impurities by IR, HRMS and NMR. The impurities were proposed based on the molecular weight
revealed by LC-MS, and confirmed by their synthesis followed by spectroscopic analysis; (3) develop
an effective and sensitive HPLC method to separate and quantify all the related substances of LDX. To
our knowledge, this is the first comprehensive study on process-related and degradation impurities in
LDX including their characterization and probable mechanisms of formation, and on development of
an effective HPLC method to separate and quantify them.
2. Results and Discussion
2.1. Detection of Process-Related Impurities and Forced Degradation of LDX
After analysis of different laboratory batches of LDX, process-related impurities were detected in
the range of 0.05–1.61%. The HPLC method described in Section 3.2 was used to obtain a typical LC-UV
chromatogram of a bulk drug sample of LDX, presenting eleven peaks (the retention time of Imp-B
and Imp-C was the same due to their being enantiomers), Imp-H (RT = 8.667, relative retention time
(RRT) = 0.725); Imp-L (RT = 9.908, RRT = 0.828); Imp-M (RT = 10.305, RRT = 0.862); Imp-E (RT = 10.728
RRT = 0.897); Imp-D (RT = 11.145, RRT = 0.932); Imp-B and Imp-C (RT = 13.467, RRT = 1.126); Imp-A
(RT = 14.025, RRT = 1.173); Imp-K (RT = 25.740, RRT = 2.153); Imp-G (RT = 26.907, RRT = 2.251), Imp-F
,

Molecules 2018, 23, 3125
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(RT = 28.440, RRT = 2.379); Imp-J (RT = 36.838, RRT = 3.082), which were shown in Figure 2. Moreover,
the LDX samples were analyzed by LC-MS and the molecular weights were 135.1 (Imp-A), 263.2
(Imp-B and Imp-C), 391.2 (Imp-D), 391.2 (Imp-E), 363.2 (Imp-F), 363.2 (Imp-G), 519.3 (Imp-H), 463.3
(Imp-J), 163.1 (Imp-K), 249.2 (Imp-L) and 279.2 (Imp-M), respectively (Figure S5).
During the course of degradation studies (under acidic, thermal and photolytic conditions),
no significant change in the sample purity was observed. However, three degraded products
(Imp-A, -B and -C) under alkaline and one degradation product (Imp-M) under oxidative conditions
were detected.
Figure 2. Typical HPLC chromatogram of LDX spiked with its impurities.
2.2. Impurity Preparation and Structural Confirmation
All twelve LDX impurities were synthesized in our laboratory and further confirmed by IR, HRMS,
NMR, and MS/MS spectroscopy. The HRMS data and carbon atom numbering scheme were shown in
Table 1 and the 1H-NMR and 13C-NMR spectral data of the impurities were shown in Tables 2 and 3
,
respectively. There were detailed descriptions on the structural characterization of (1S)-1-phenyl
propan-2-amine (Imp-A) [ ], (2S)-2,6-di-((tert-butoxycarbonyl)amino)-N-[(1S)-1-methyl-2-phenyl
8,9
ethyl]hexanamide (Imp-J) [10] and N-[(1S)-1-methyl-2-phenylethyl]formamide (Imp-K) [11] in the
literature. Accordingly, the structures of Imp-A, Imp-J and Imp-K were confirmed by comparison with
published spectral data. All the relevant spectral data for structural confirmation are shown in the
Supporting Information.
2.2.1. Structural Elucidation and Control Strategy of Imp-B and Imp-C
Imp-B and Imp-C originated from the enantiomers of two different starting materials 2, 3
The synthetic route of Imp-B and Imp-C were consistent with LDX (Figure 3), except that L-lysine (
was replaced by D-lysine (2a) or (S)-1-phenylethanamine ( ) was replaced by (R)-1-phenylethanamine
3a). Imp-B and Imp-C, were obtained as white solids and their HPLC purities were 98.63% and 96.71%,
.
2)
3
(
respectively. The HRMS of Imp-B and Imp-C showed an [M + H]+ at m/z 264.2071 and 264.2069,
respectively, suggesting the same elemental composition of C15H26N3O (Table 1) as LDX. Imp-B and
Imp-C were at the same position in reversed-phase liquid chromatography but were displayed as two
different peaks (RT = 12.740 min and 14.288 min) in normal-phase chromatography (in Supporting
Information Figure S6), which indicated that they were isomers instead of an identical compound.
Specific rotations of Imp-B and Imp-C were +6.512 and
−
6.847, respectively, further supporting that
Imp-B and Imp-C, with identical molecular formulae, were diastereoisomers of LDX. Detailed 1H-NMR
spectral data were given in Table 2. The control strategy of Imp-B and Imp-C was to minimize the
isomers of intermediate
8 by recrystallization (acetone:n-heptane = 1:10, v/v). Furthermore, by means

Molecules 2018, 23, 3125
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of recrystallization of LDX, Imp-B and Imp-C were easily removed leaving less than 0.1% content in
the bulk drug.
Table 1. Retention time, HRMS and structures of LDX and its impurities.
HRMS
Compound
RRT
1.00
1.17
1.12
Structure
Source
[M + H] +
Chemical Formula
LDX
Imp-A
Imp-B
264.2076
C15H26N3O
Target compound
136.1121
264.2071
C9H13
N
Process and alkaline degradation
Process and alkaline degradation
C15H26N3O
C15H26N3O
Imp-C
Imp-D
1.12
0.93
264.2069
392.3205
Process and alkaline degradation
Process
C21H38N5O2
Imp-E
Imp-F
0.89
2.38
392.3207
364.2595
C21H38N5O2
C20H34N3O3
Process
Process
Imp-G
Imp-H
Imp-J
2.25
0.72
3.08
364.2590
520.3975
464.3118
C20H34N3O3
C27H50N7O3
C25H41N3O5
Process
Process
Process
186.0889 (M +
Na+)
Imp-K
Imp-L
2.15
0.82
C10H13NO
C14H24N3O
Process
Process
250.1908
280.2020
Imp-M
0.86
C15H26N3O2
Oxidative degradation

Molecules 2018, 23, 3125
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1
Table 2. H-NMR assignment for LDX and its impurities.
Position
LDX
Imp-B
Imp-C
Imp-D
Imp-E
Imp-F
Imp-G
Imp-H
Imp-L
Imp-M
3.66–3.63
(t,1H)
1.48–1.40
(m (c),4H)
1.03–0.92
(m,2H)
1.48–1.44
(m,4H)
2.78–2.73
(m,2H)
3.67–3.63
(t,1H)
3.67–3.63
(t,1H)
4.22–4.18
(t,1H)
3.63–3.61
(t,1H)
3.10–3.07
(t,1H)
3.99–3.95
(t,1H)
4.20–4.12
(dt,1H)
1.49–1.43
(m,2H)
1.13–1.11
(d,2H)
1.38–1.34
(m,6H)
3.06–3.01
(m,2H)
4.02–3.95
(m,1H)
1.07
4.93–4.88
(m,1H)
1.53–1.46
(m,2H)
1.25–1.14
(m,2H)
1.69–1.66
(m,2H)
3.65–3.63
(t,1H)
2
3
1.76–1.67
(dd (d),2H
1.35–1.26
(m,2H)
1.59–1.47
(m,2H)
2.78–2.71
(m,2H)
3.97–3.93
(m,1H)
1.03
1.76–1.68
(dd,2H)
1.35–1.26
(m,2H)
1.59–1.47
(m,2H)
2.78–2.70
(m,2H)
3.97–3.93
(m,1H)
1.02
1.79–1.69
(m,2H)
1.39–1.31
(m,2H)
1.63–1.48
(m,6H)
2.82–2.76
(m,4H)
3.97–3.92
(m,1H)
1.01
1.75–1.68
(m,2H)
1.38–1.27
(m,4H)
1.49–1.45
(m,2H)
3.03–2.98
(m,2H)
4.15–4.07
(m,1H)
1.14
1.47,1.30
(m,2H)
1.24–1.17
(m,2H)
1.34–1.26
(m,2H)
2.89–2.84
(dd,2H)
3.93–4.03
(m,1H)
1.03
1.54–1.46
(m,2H)
1.28–1.23
(dd,2H)
1.43–1.34
(m,2H)
2.69–2.61
(m,2H)
4.32–4.32
(m,1H)
1.15
1.54–1.46
(dd,2H),
1.08–0.86
(m,2H).
1.44–1.39
(m,2H),
2.68–2.61
(m,2H),
4.20–4.09
(m,1H),
1.11–1.10
(d,3H),
2.72,2.56
(dd,2H)
7.04(d,1H),
7.01(t,H)
6.69(d,1H),
6.78(t,H)
4
5
2.67–2.61
(dd,2H)
6
4.16–4.04
(m,1H)
7
–
1.13
1.38
(d,3H)
3.79
8
(d (b),3H)
2.65,2.75
(dd,2H)
7.24–7.18
(m,3H)
(d,3H)
(d,3H)
(d,3H)
(d,3H)
(d,3H)
(d,3H)
(d,3H)
2.69–2.67
(d,2H)
2.62,2.82
(dd,2H)
7.23–7.17
(m,3H)
7.32–7.26
(m,2H)
7.23–7.17
(m,3H)
8.43–8.37
(d,1H)
8.20–8.10
(s,3H)
7.82–7.72
(s,3H)
2.62,2.82
(dd,2H)
7.23–7.16
(m,3H)
7.32–7.25
(m,2H)
7.23–7.16
(m,3H)
8.43–8.37
(d,1H)
8.20–8.10
(s,3H)
7.82–7.72
(s,3H)
2.62,2.79
(dd,2H)
7.23–7.17
(m,3H)
7.31–7.26
(m,2H)
7.23–7.17
(m,3H)
8.01–7.99
(d,1H)
8.65–8.63
(d,1H)
7.78
(s,3H)
2.39
(s,9H)
3.88–3.83
(t,1H)
1.63–1.48
(m,6H)
1.25
2.68,2.76
(dd,2H)
7.23–7.18
(m,3H)
7.30–7.26
(m,2H)
7.23–7.18
(m,3H)
8.43–8.40
(t,1H)
8.10–8.09
(d,3H)
8.33–8.30
(d,1H)
2.38
(s,9H)
3.72–3.69
(t,1H)
1.60–1.52
(dd,2H)
1.04,0.93
(m,2H).
1.38–1.27
(m,4H)
2.79–2.74
(dd,2H)
7.70
2.77,2.63
(dd,2H)
7.20–7.17
(m,3H)
7.29–7.26
(m,2H)
7.20–7.17
(m,3H)
7.80–7.77
(d,1H)
2.84–2.71
(dd,2H)
7.24–7.18
(m,3H)
7.31–7.28
(m,2H)
7.24–7.18
(m,3H)
5.15
9
(t,1H)
7.20–7.19
(m,3H)
7.30–7.23
(m,2H)
7.20–7.19
(m,3H)
7.99–7.97
(d,1H)
8.43–8.42
(d,2H)
8.43–8.42
(d,2H)
7.34–7.28
(m,4H)
7.34–7.28
(m,4H)
7.34–7.28
(m,4H)
8.95–8.93
(d,1H)
8.18–8.13
(m,3H)
7.78
11,13
12,14
15
7.31–7.26
(m,2H)
7.24–7.18
(m,3H)
8.38–8.32
(d,1H)
8.15–7.56
(s,6H)
–
8.27
(d,1H)
8.08
(s,2H)
7.86
(s,2H)
9.43
(s,1H)
16
(d,1H)
6.16
(s,1H)
17
–
8.15–7.56
(s (a),6H)
2.39
6.75–6.73
(t,1H)
18
–
(s,3H)
2.45
(s,6H)
2.42
(s,6H)
2.42
(s,6H)
2.45
19
–
–
(s,6H)
(s,12H)
3.82–3.73
(t,1H)
1.72–1.70
(d,4H)
1.38–1.34
(m,6H)
1.62–1.53
(dd,4H)
2.84–2.71
(dd,4H)
7.75
1.37
(s,9H)
1.37
(s,9H)
1.37
1.45
(s,9H)
1.45
(s,9H)
1.45
21,29
22,31
23,32
24,33
25,34
26,(35)
27,(30)
(d,2H)
(s,9H)
(s,9H)
1.63–1.48
(m,6H)
2.82–2.76
(m,4H)
7.78
(s,3H)
8.14
(s,3H)
7.99
(d,3H)
(s,6H)
8.09
(s,6H)
(s,3H)
(a)
Single; (b) Double; (c) Multiple; (d) Doublet doublet.
Table 3. 13C-NMR assignment for LDX and its impurities.
LDX
Imp–D
Imp–E
Imp–F
Imp–G
Imp–H
Imp–L
Imp–M
Position
δC DEPT
δC DEPT
δC DEPT
δC DEPT
δC DEPT
δC DEPT
δC DEPT
δC DEPT
1
2
3
4
5
6
7
8
9
167.41–
168.88–
167.87–
174.25–
54.98 CH
35.00 CH2
22.88 CH2
29.89 CH2
40.25 CH2
46.00 CH
20.54 CH3
42.29 CH2
139.43 C
128.55 CH
129.62 CH
126.45 CH
129.62 CH
128.55 CH
156.03–
174.17–
54.97 CH
34.55 CH2
22.72 CH2
29.83 CH2
40.09 CH2
45.83 CH
20.23 CH3
42.62 CH2
138.25 C
128.30 CH
129.38 CH
126.37 CH
129.38 CH
128.30 CH
156.10–
170.61–
53.34 CH
28.97 CH2
22.96 CH2
26.72 CH2
32.14 CH2
46.54 CH
21.02 CH3
42.19 CH2
139.51 C
128.46 CH
129.59 CH
126.47 CH
129.55 CH
128.49 CH
40.16 CH3
168.63–
52.53 CH
30.90 CH2
21.65 CH2
26.78 CH2
38.97 CH2
168.69–
52.25 CH
30.84 CH2
21.33 CH2
167.96–
52.33 CH
30.78 CH
22.74 CH2
26.66 CH2
38.95 CH2
–
21.46 CH3
48.91 CH
144.61 C
127.32 CH
128.78 CH
126.37 CH
128.78 CH
127.32 CH
40.11 CH3
167.69–
52.47 CH
30.88 CH2
21.27 CH2
27.01 CH2
38.93 CH2
45.31 CH
21.47 CH3
36.97 CH2
125.37 C
131.29 CH
118.94 CH
127.73 CH
115.28 CH
155.85 C
–
52.02 CH
30.38 CH2
20.73 CH2
26.38 CH2
38.53 CH2
46.45 CH
20.83 CH3
41.77 CH2
138.99 C
128.13 CH
129.20 CH
126.18 CH
129.20 CH
128.13 CH
39.80 CH3
53.35 CH
30.89 CH2
21.58 CH2
26.72 CH2
38.98 CH2
46.66 CH
20.17 CH3
41.99 CH2
139.43 C
128.62 CH
129.61 CH
128.62 CH
129.61 CH
128.62 CH
40.07 CH3
170.61–
52.61 CH
30.88 CH2
21.57 CH2
26.78 CH2
38.90 CH2
46.84 CH
21.22 CH3
42.17 CH2
139.34 C
128.50 CH
129.57 CH
126.56 CH
129.57 CH
128.50 CH
40.13 CH3
168.67–
10
11
12
13
14
15
19
20
21
22
23
24,33
25,34
28
29
31
32
77.73 C
79.07 C
52.47 CH
31.98 CH2
22.63 CH2
26.86 CH2
39.10 CH2
52.53 CH
31.07 CH2
21.64 CH2
28.83 CH2
38.96 CH2
28.74 CH3
28.74 CH3
28.74 CH3
28.42 CH3
28.42 CH3
28.42 CH3
Note: 13C-NMR assignment for Imp-B and Imp-C was identical to LDX.

Molecules 2018, 23, 3125
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Figure 3. Eleven routes for the formation of LDX and its impurities.
2.2.2. Structural Elucidation and Control Strategy of Imp-F and Imp-G
Both of the protonated molecular ions for Imp-F and Imp-G, were obtained at an [M + H]+ of
m/z 364.2 (Figure S5), which was 100 a.m.u. more than that of LDX. The 100 may correspond to
one t-butyloxy carbonyl (Boc) moiety and thus we speculated that the two impurities were possibly
(2S)-2-amino-6-((tert-butoxycarbonyl)amino)-N-[(1S)-1-methyl-2-phenylethyl]hexan-amide (Imp-F)
and (2S)-2-((tert-butoxycarbonyl)amino)-6-amino-N-[(1S)-1-methyl-2-phenyl-ethyl]hexanamide (Imp-
G), respectively. According to the synthetic route (Figure 3), Imp-F was obtained as a colorless oil with
97.63% HPLC purity while Imp-G was obtained as a white solid with 98.63% HPLC purity.
The HRMS of impurities F and G showed an [M + H]+ at m/z 364.2595 and 364.2590, respectively,
suggesting an identical elemental composition of C20H34N3O3 (Table 1). The structures were further
1
confirmed by the IR, H-NMR, 13C-NMR, and DEPT spectra. Both of these impurities had one
additional t-butyloxycarbonyl compared with LDX. In the 13C-NMR of Imp-F, the additional Boc group
was deshielded to δC20 = 77.73 ppm and δC21 = 28.74 ppm. In the 1H-NMR, the chemical shift of the
additional Boc was deshielded to δH21 = 1.37 ppm. The NMR spectrum of Imp-G was similar to that
of Imp-F, except that H-2 was appeared at a lower field of the 1H-NMR spectrum (chemical shift of
δH = 3.98 ppm) that impurity G, which was affected by the acyl-amino groups at C1 and C19. This
phenomenon also ocurrs between 2b and 2c (Supporting Information Figure S7). Detailed information
about the 1H-NMR and 13C-NMR spectra can be seen in Tables 2 and 3. To the best of our knowledge,
this is the first report of all the spectroscopic data of Imp-F and Imp-G.

Molecules 2018, 23, 3125
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There were two ways whereby Imp-F and Imp-G can be formed in the bulk drug. First,
intermediate may contain 2b and 2c if the amino protection was not complete during its synthesis,
5
affording, respectively, Imp-F and Imp-G. Second, the presence of Imp-J in LDX drug substance
indicated that Imp-F and Imp-G were also likely formed due to the incomplete de-Boc in the final
step of LDX. Accordingly, the control strategy of Imp-F and Imp-G was to increase the equivalents
of (Boc)2O to 2.2 during the amino protection, so that L-lysine reacted completely as far as possible,
thereby reducing the content of 2b and 2c. Moreover, the amount of methanesulfonic acid was
increased to 5 equivalents in the last step to ensure he complete deprotection. The content of Imp-F,
Imp-G and Imp-J can thus be reduced to below 0.1% after recrystallization.
2.2.3. Structural Elucidation and Control Strategy of Imp-D and Imp-E
On-line LC-MS spectra of Imp-D and Imp-E in LDX suggested that they were
likely (2S)-2-lysyl-6-amino-N-[(1S)-1-methyl-2-phenylethyl]hexanamide (Imp-D), and (2S)-2-amino
-6-lysyl-N-[(1S)-1-methyl-2-phenylethyl]hexanamide (Imp-E), both of which were colorless oils with
≥
96% HPLC purity. The HRMS of impurity D and E revealed their [M + H]+ at m/z 392.3205 and
392.3207, respectively, suggesting that they share the same elemental composition of C21H38N5O2
(Table 1). Their structures were further confirmed by IR, H-NMR, 13C-NMR, DEPT, HSQC and
1
HMBC spectral data. The 1H-NMR spectra of Imp-D and Imp-E showed 17 signals, corresponding to
49 protons, which were consistent with the molecular structures of Imp-D and Imp-E. Both of them had
an additional L-lysine on different amino groups (H17 and H18) compared with LDX. In the 13C-NMR
spectrum of Imp-E, the chemical shift of the additional L-lysine (C20, C21, C22, C23, C24, C25) was
deshielded to δC = 168.64, 52.53, 31.07, 21.64, 28.83, 38.96 ppm, respectively. The 13C-NMR spectrum
of Imp-D was similar to that of Imp-E. In the 1H-NMR spectrum of Imp-D (Supporting Information
Figure S7, D-1), affected by the two carbonyl groups (C1 and C20), the H-2 proton appeared at a lower
field (chemical shift of δH2 = 4.20 ppm) while its chemical shift is 3.60 ppm in the 1H-NMR spectrum of
Imp-E (Figure S7, E-1). Besides, compared with the H-6 of Imp-D (δH6 = 2.80 ppm), the H-6 of Imp-E,
affected by the acyl-amino group (C20), shifted to a lower field (δH6 = 3.02 ppm). The HSQC spectrum
provided further evidence for the difference on the structures of Imp-D and Imp-E (Figures S7, D-5,
E-5). In the HMBC spectrum of Imp-D (Figure S7, D-6), H-2 was correlated with C1 and C20, but the
key long-range correlation between H-2 and C20 in Imp-E was not existed (Figure S7, E-6). In the
meantime, there was no correlation between H-6 and C20 in Imp-D (Figure S7, D-6), while the H-6
was correlated with C20 in Imp-E (Figure S7, E-6). The correlation peaks of two-dimensional NMR
spectra indicated that Imp-D and Imp-E were not the same compound, but rather positional isomers.
The detailed 1H-NMR and 13C-NMR spectra information can be seen in Tables 2 and 3. The HRMS
and NMR spectra of the two impurities have never been reported in the literature.
In order to control the amount of Imp-D and Imp-E, we decreased the content of 2b and 2c by
optimizing the process parameters in the amino protection step. In addition, intermediate
8 was
recrystallized (acetone:n-heptane = 1:10, v/v) to reduce the precursors of Imp-D and Imp-E. As a result,
the content of the two impurities in LDX were eliminated to below 0.05%.
2.2.4. Structural Elucidation and Control Strategy of Imp-H
Inspired by the formation of Imp-D and Imp-E and a [M + H]+ m/z 520.3 peak for Imp-H
(Figure S5), we speculated that Imp-H was (2S)-2,6-di-(lysyl)-amino-N-[(1S)-1-methyl-2-phenylethyl]
hexanamide. Imp-H was obtained as a white solid and its HPLC purity was found to be 99.61%.
By comparison of retention times in HPLC, we found that the quantity of Imp-H was about 0.05% in
the bulk drug (Figure 2). The HRMS of impurity H revealed an [M+H] + at m/z 520.3975, suggesting an
elemental composition of C27H50N7O3 (Table 1). Besides, the fragments 503.4, 392.3, 385.3, 264.2, 257.2,
129.1 appeared in the MS/MS spectrum of Imp-H, which supported the proposed molecular structure
(Figure 4). The structure was further confirmed by IR, 1D NMR (1H, 13C, DEPT) and 2D NMR (COSY,
HSQC, HMBC) spectral data. The IR spectrum displayed characteristic absorptions at 3431.0, 1671.4,

Molecules 2018, 23, 3125
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1555.5 and 1192.9/cm which were indicatives of an amino (N-H) stretching vibration, a C=O stretching
vibration, an N-H bending vibration, and a C-N stretching vibration, respectively. Imp-H had two
additional acyl-amino groups (-CONH) and two additional amino-groups (-NH2) compared with LDX.
The chemical shift of the additional active hydrogens (H17, H18) were deshielded to δH = 7.8–8.5 ppm
1
compared with the H-NMR spectrum of LDX. The additional amino groups were assigned to be
H-27, H-30, H-26, H-35 on the basis of the HMBC spectrum in the Supporting Information (Figure S7)
showing correlations of H-27, H-30 (δH =8.08 ppm) with C-20, C-28 (δC = 168.69, 168.63 ppm) and
correlations of C-20, C-28 (δC = 168.69, 168.63 ppm) with H-17, H-18 (δH =8.43–8.42 ppm) (Figure S7,
H-5). The above results indicated that Imp-H had two additional L-lysines compared with LDX.
Furthermore, the H-2 and H-6 signals appeared at a lower field in the 1H-NMR spectrum (chemical
shift of δH2 = 4.18 ppm and δH2 = 3.04 ppm, respectively) of impurity H (Figure S7), which indicated
that the amino-group (-NH2) was transformed to an acylmino (-CONH). The COSY spectrum showed
correlation of H-17 (δH = 8.42 ppm) with H-2 (δH = 4.18 ppm) and correlation of H-18 (δH = 8.43 ppm)
with the methylene H-6 (δH = 3.04 ppm). In the meantime, there were twelve more carbon atoms in
Imp-H than in LDX, and the chemical shifts of δC28 = 168.69 ppm, δC20 = 168.63 ppm and δC1 = 170.61
ppm in the 13C-NMR spectrum provided further evidence for the existence of amides. The assignment
of 1H- and 13C-NMR signals was performed for Imp-H on the basis of the 1H-, 13C- and 2D NMR data
in Tables 2 and 3. Further detailed information of the HSQC, HMBC and COSY spectra of Imp-H
can be seen in Figure S7. To our knowledge, this compound is reported for the first time. The control
strategy of Imp-H is identical to that of Imp-D and Imp-E.
2.2.5. Structural Elucidation and Control Strategy of Imp-L
The synthetic route of (2S)-2,6-di-amino-N-((1S)-phenylethyl) hexanamide (Imp-L) was similar to
that of LDX (Figure1), except that Imp-A was replaced by (S)-1-phenylethylamine (3) in the amide
condensation step. Imp-L was obtained as a white solid and its HPLC purity was found to be 98.63%.
The HRMS of Imp-L revealed an [M + H]+ at m/z 250.1908, which suggested an elemental composition
of C14H24N3O (Table 1). Compared with LDX, Imp-L was missing a -CH2 group. In the 1H-NMR
spectrum, no benzyl (H7) was found at δH = 2.6–2.9 ppm and the chemical shift of H8 was deshielded
from 1.14–1.15 ppm to 1.37–1.39 ppm. Detailed information about the 1H-NMR and 13C-NMR spectra
is given in Tables 2 and 3. The IR spectrum of Imp-L displayed characteristic absorptions at 3449.5,
1597.1 and 1198.5/cm which were indicative of an amino (N-H) stretching vibration, a C=O stretching
vibration and a C-N stretching vibration, respectively.
The residue of (S)-1-phenylethanamine (
strategy of Imp-L was to make react as completely as possible in the reductive amination reaction.
Thus, the equivalent ratio of (S)-1-phenylethanamine ( ) and phenylacetone ( ) was set to 1:1.1. On the
other hand, the chemical and optical purity of intermediate were improved by salt formation with
hydrochloric acid, thereby reducing the production of Imp-L from the source.
3) in intermediate 7 led to Imp-L. Hence, the control
3
3
4
6

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Figure 4. Cont.

Molecules 2018, 23, 3125
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Figure 4. MS/MS spectra and plausible fragments of LDX (a), Imp-H (b) and Imp-M (c).
2.2.6. Structural Elucidation and Control Strategy of Imp-M
Oxidative degradation was performed in 5% H2O2 at room temperature in the dark for 4h.
Considering that the purpose of the degradation experiment is to provide recommendations for
transport and storage of drugs, we focused on the impurity with the maximum content under this
oxidative condition. The on-line LC-MS spectrum indicated that the molecular weight of the major
degradation product was 279.2 (Figure S5), 16 more than LDX. The HRMS of impurity M showed an
[M + H]+ at m/z 280.2020, suggesting an elemental composition of C15H26N3O2 (Table 1). Furthermore,
there was only four hydrogen atoms on the benzene ring accorded with the 1H-NMR spectrum. In other
words, impurity M had more than one substituent group on the benzene ring and not the two primary
amines of LDX. It was supposed that the two primary amines had formed salts with methanesulfonic
acid, making them more stable in hydrogen peroxide. Furthermore, the identical oxidative experiment
had been conducted with the free base of LDX, but the degraded products showed different retention
time with Imp-M in HPLC. Moreover, it was reported that hydrogen peroxide with strong acid or
Lewis acid converted benzene and alkylbenzenes into their hydroxylated products [12]. On the basis of
molecular weight (279.2), we speculated that the additional group was a hydroxyl group. In addition,
the 1H-NMR spectrum showed that there were four different kinds of hydrogen on the benzene ring in
the low field. Thus, we excluded the para-hydroxyl degradant. Moreover, the splitting of these four
kinds of hydrogen are double and triple peaks, but not single, which indicated the hydroxyl group was
not located in the meta-position. The HMBC spectrum of Imp-M in Supporting Information Figure S7
showed correlations of the additional hydroxyl group (H-19) (δH =9.43 ppm) with C-10, C-14 and C-15
(δC = 125.30, 115.25, 155.85 ppm) and correlations of H-9 (δH = 2.73, 2.56 ppm, dd) with C-10, C-11 and
C-15 (δC = 125.30, 131.29, 155.85 ppm). The above results supported that the additional OH was located
in the ortho-position. The structure was further confirmed by 13C-NMR and DEPT. In the 13C-NMR,
the carbon atom connecting to the additional OH shifted to the low field (δC15 = 155.85 ppm) compared
to that of LDX. In the meantime, the DEPT spectrum showed that only four carbons appeared in the
aromatic region (110–160 ppm), and the C-15 (δC15 = 155.85 ppm) disappeared, which confirmed again
that the OH was on the benzene ring. The HSQC spectrum of Imp-M (Figure S7, M-5) showed that
there was no hydrogen atom correlated with C-10 and C-15 (δC-10 = 1125.30 ppm, δC-15 = 155.85 ppm),
which provided further evidence for the above conclusion. Based on the abovementioned spectral data,
the new compound was identified as (2S)-2,6-diamino-N-[(1S)-1-methyl-2-(2-hydroxyphenyl) ethyl]

Molecules 2018, 23, 3125
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hexanamide. Fragments 263.2, 246.2, 152.1, 135.1, 129.1, 84.1 were visible in the MS/MS spectrum of
Imp-M, which further supports the proposed molecular structure (Figure 4). The assignment of 1H-
and 13C-NMR signals was completed by means of COSY, HSQC and HMBC spectroscopic data sets
(Figure S7). The detailed information about the 1H-NMR, 13C-NMR and DEPT spectra can be seen in
Tables 2 and 3. This novel degradation product has not yet been disclosed in any other published work.
2.3. Possible Mechanisms for Formation of the Impurities
Taking into account the synthetic process of LDX in combination with some published research,
we proposed eleven possible routes for the formation of the twelve impurities (process-related and
degradation) (Figure 3). Imp-A and Imp-J were the residues of intermediate
in the synthetic process of LDX in route 1. In routes 2 and 3, both intermediate
L-lysine ( ) contained trace amounts of enantiomers A-1 and 2a The synthetic routes of
Imp-B and Imp-C were consistent with that of LDX (Figure 1), except that L-lysine ( ) was
replaced by D-lysine (2a) while intermediate was replaced by (R)-1-phenylpropan-2-amine
A-1). On the other hand, LDX can produce Imp-B or Imp-C in alkaline condition. In routes 4
and 5, intermediate may contain (2S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanoic acid (2b
7
and intermediate
8
7
and
2
.
2
7
(
5
)
and (2S)-6-amino-2-((tert-butoxycarbonyl)-amino)hexanoic acid (2c) as impurities when the amino
protection was not completed during its synthesis, affording, respectively, Imp-F and Imp-G which,
generated Imp-D (route 6) and Imp-E (route 7) after sequentially reacting with intermediate
both underwent the same reaction that gave LDX. In route 8, intermediate may contain the residue
of L-lysine ( ) in its synthesis process, affording Imp-H with the same reaction that gave LDX in
the last step. In route 9, during the debenzyl reaction, excessive amounts of ammonium formate
may continuously react with intermediate , affording Imp-K as a residue in LDX. In route 10, as an
impurity, might exist in intermediate , and Imp-L was obtained by the same reaction for LDX. In
5, and
5
2
7
3
7
route 11, Imp-M was produced under oxidative condition.
2.4. Optimization of the HPLC-UV Method
According to the foregoing analysis, twelve impurities were detected and successfully identified
by LC-MS, HRMS, NMR and IR spectroscopy. Initially, different types of HPLC columns, such as
Thermo Accucore XL C8 (150
column and YMC-Pack ODS-AQ (250
of separating LDX and its impurities was evaluated mainly through the performance characteristics of
the columns. The best resolution was obtained on the discovery YMC-Pack ODS-AQ column which
was thereafter used for further optimization of the method.
×
4.6 mm, 4
µ
m) column, Thermo Syncronis C18 (250
×
4.6 mm
×
5 µm)
×
4.6 mm
×
5 µm) were tested to analyze LDX. The capability
Different mobile phase conditions and gradient progress were tested together to develop a
selective separation method. We used a variety of organic acids and the tailing peak was found to
appear when trifluoroacetic acid was used as the mobile phase. Fortunately, better shape symmetrical
peaks were obtained with methanesulfonic acid. In the meantime, the addition of 0.1% methanesulfonic
acid to acetonitrile improved the baseline fluctuation. The separation of these impurities was not
satisfactory by a continuous gradient elution program. The initial gradient elution condition was as
follows: 0–10 min, linear from 5% to 20% B, however, the polar impurities (D, E, L, M) cannot be well
separated under this condition. For the separation of Imp-D, Imp-E, Imp-L and Imp-M, the gradient
profile was optimized. On the one hand, we reduced the slope of B increase (0–15 min, linear from
3% to 20% B). Alternatively, the separation can be improved by reducing the initial proportion of the
organic phase to 3%. The method was initially optimized by comparing the separation of related
substance, shape symmetrical peaks of LDX and its impurities, and then by optimizing the effect of
column types, mobile phase and gradient elution mode afterwards shown in Section 2.2.

Molecules 2018, 23, 3125
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2.5. Validation of the HPLC-UV Method
The HPLC method, used to identify the related substances in LDX bulk drug, was validated
in terms of the linearity, accuracy, precision, limit of quantitation (LOQ), limit of detection (LOD),
robustness and system suitability. The validation was in accordance with ICH Q2 guideline [13] and
the details are shown in Tables 4 and 5.
Table 4. Summary of method validation.
System Suitability
Linearity
Slope
Sensitivity
Compound
RRT a
SF c
Range (µg/mL)
R e
Intercept
LOD g (µg/mL)
PC b
R d
CF f
LOQ h (µg/mL)
LDX
Imp-A
Imp-B
Imp-C
−Imp-D
Imp-E
Imp-F
Imp-G
Imp-H
Imp-J
-
19755
105353
121354
125659
98972
118365
749255
864920
46840
1.28
1.15
1.02
1.03
1.09
1.14
1.63
1.16
1.28
1.14
1.12
1.02
1.17
6.09
66.28
3.50
3.50
3.33
3.19
12.44
80.95
8.53
-
0.5100–20.4000
0.5025–20.1000
0.5110–20.4400
0.5047–20.1900
0.5160–20.6400
0.5135–20.5400
0.5022–20.0900
0.5070–20.2800
0.5028–20.1900
0.5080–20.3200
0.5105–20.256
0.5180–20.7200
0.5240–20.9592
0.9999 0.1461
1.0000 0.2271
1.0000 0.1262
1.0000 0.1119
1.0000 0.1112
1.0000 0.0836
1.0000 0.1700
1.0000 0.1641
1.0000 0.1524
0.9998 0.1269
0.9999 0.1389
1.0000 0.1159
1.0000 0.1614
−0.0055
−0.0276
−0.0141
−0.0010
−0.0079
−0.0013
−0.0061
−0.0119
−0.0085
0.0110
-
0.3060
0.3028
0.3010
0.3026
0.3035
0.3041
0.3013
0.3042
0.3028
0.3028
0.3036
0.3041
0.3060
0.5100
0.5025
0.5105
0.5105
0.5070
0.5051
0.5023
0.5070
0.5062
0.5041
0.5075
0.5180
0.5140
1.17
1.12
1.12
0.93
0.89
2.38
2.25
0.725
3.08
2.15
0.83
0.86
0.61
1.10
1.31
1.04
1.66
0.86
0.89
0.92
1.15
1.32
1.19
0.91
28465
322224
90222
Imp-K
Imp-L
Imp-M
7.00
8.53
3.40
0.0105
−0.0056
114119
−0.0011
a Relative retention time; b (USP) plate count; c Symmetry factor; d (USP) resolution; e Correlation factor. f Calibration
response factor. (S/N ≥ 3). (S/N ≥10).
g
h
Table 5. Summary of accuracy.
Impurity 0.05%–1% 0.05%–2% 0.05%–3% 0.10%–1% 0.10%–2% 0.10%–3% 0.15%–1% 0.15%–2% 0.15%–3%
Mean
RSD (n = 9)
Imp-A
Imp-B
Imp-C
Imp-D
Imp-E
Imp-F
Imp-G
Imp-H
Imp-J
91.8
102.6
92.8
99.7
99.1
103.9
101.5
99.5
92.7
99.7
91.4
97.8
90.2
105.1
90.4
96.3
101.0
96.6
102.4
98.7
95.4
103.5
90.6
98.5
92.6
100.2
91.2
95.8
102.7
97.1
100.3
102.5
93.9
101.6
101.2
93.2
89.6
100.9
100.6
97.7
101.6
103.3
103.3
101.8
95.0
92.7
91.3
98.7
96.3
96.5
105.6
96.6
98.9
96.2
99.5
95.5
94.4
94.1
97.3
99.5
97.6
95.7
99.3
102.6
103.5
93.5
96.4
94.8
97.6
92.0
92.6
97.4
94.1
103.5
98.8
95.8
96.9
91.0
105.1
96.7
91.3
92.5
90.8
102.4
97.0
103.4
100.3
100.9
99.5
94.1
90.5
98.5
93.1
102.6
98.1
100.5
102.1
94.0
104.2
94.9
92.0
96.8
96.8
1.83
5.82
4.44
2.04
2.91
3.19
2.59
2.16
2.50
2.90
3.69
2.62
96.4
100.6
100.3
100.4
100.3
94.5
101.3
94.5
95.1
99.4
103.6
90.0
Imp-K
Imp-L
Imp-M
98.7
95.7
95.1
95.5
92.7
2.5.1. System Suitability
In order to obtain a satisfactory performance using the analytical method, a system suitability
test was carried out before each run. The results showed that the United States Pharmacopoeia (USP)
theoretical plates of LDX and its impurities were greater than 19755, the USP resolution between any
two compounds was greater than 3.19, and the peak asymmetry for all the analytes was between 1.02
and 1.28 (Table 4). The HPLC chromatogram of the separation of LDX and its impurities can be seen in
Figure 2.
2.5.2. Linearity, LOD, and LOQ
Using the least squares method, linear regression analysis of the response values of sample
solutions with different concentrations and the corresponding concentration was carried out to
calculate the slope and intercept. The measurements indicated that the response value and
concentration had a positive linear relationship over the concentration range of 0.50–20.00
µg/mL.
The LOQ solution (0.50 g/mL), equivalent to 0.05% of the LDX sample solution, was prepared and
µ
used to calculate the (S/N) of LDX and its twelve impurities. S/N of LDX and its impurities was
greater than 10, and the LOQ of the method was 0.05% while the minimum quantitative concentration
was 0.50 µg/mL. Using the same injection, the calculated LOD of the method was 0.02% and the
S/N of LDX and its impurities was 3:1. The LOQ level by injecting six individual preparations and
calculating the percentage RSD of the areas. The results were shown in Table 4.

Molecules 2018, 23, 3125
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2.5.3. Accuracy, Precision, and Robustness
Recovery and RSD values of sample solution at concentration levels of 0.05%, 0.10%, and 0.15%
were measured in triplicate after the addition of a certain amount of the twelve impurities to LDX
test solutions (1.0 mg/mL) and then the accuracy was calculated. The recovery of all the impurities
was 80%–120%, confirming the acceptable good accuracy of the method (Table 5). The precision of the
method was evaluated through parallel preparation of six individual 1.0 mg/mL LDX sample solution
for injection and calculating the RSD for each peak. The RSD of all individual impurities was not more
than 5%, indicating good precision of the method (Table 6). The robustness of the developed method
was studied by changing the column temperature (30
±
3 ◦C) flow rate (1.0
±
0.1 mL/min), detection
wavelength (215 2 nm) of the original HPLC conditions. Under different conditions, excluding the
±
isomer of impurities B and C, resolution between any two compounds was >1.5. Compared with the
original HPLC method, difference measured values of the individual impurities in the sample solution
was not more than 0.02%, suggesting excellent robustness of the method (Table 7).
Table 6. Summary of precision.
1
2
3
4
5
6
Compound
RSD (n = 6)
C (mg/mL)
1.0080
1.0225
1.0180
1.0290
1.0130
1.0095
Imp-A
Imp-B
Imp-C
Imp-D
Imp-E
0.10
0.10
0.12
0.09
0.10
0.12
0.10
0.10
0.11
0.11
0.11
0.12
0.01
0.10
0.11
0.12
0.09
0.10
0.12
0.10
0.11
0.11
0.11
0.11
0.11
0.01
0.09
0.11
0.12
0.09
0.10
0.12
0.10
0.11
0.10
0.11
0.11
0.11
0.01
0.10
0.11
0.12
0.09
0.11
0.12
0.10
0.10
0.10
0.10
0.11
0.11
0.02
0.10
0.11
0.12
0.09
0.10
0.11
0.10
0.10
0.10
0.10
0.11
0.11
0.02
0.10
0.11
0.13
0.08
0.10
0.11
0.10
0.10
0.10
0.11
0.11
0.11
0.01
4.22
2.19
3.24
1.67
1.83
1.20
1.30
1.51
2.14
1.63
1.32
1.87
2.85
Imp-F
Imp-G
Imp-H
Imp-J
Imp-K
Imp-L
Imp-M
RRT = 1.32
Table 7. Summary of robustness.
Column Temperature
Flow Rate
UV
Compound
◦
◦
◦
27
C
30
C
33 C
0.9 mL/min 1.0 mL/min 1.1 mL/min
213 nm
215 nm
217 nm
Imp-A
Imp-B
Imp-C
Imp-D
Imp-E
Imp-F
Imp-G
Imp-H
Imp-J
0.10
0.13
0.10
0.09
0.11
0.10
0.09
0.09
0.08
0.10
0.11
0.08
0.10
0.12
0.11
0.09
0.10
0.09
0.09
0.09
0.09
0.10
0.12
0.08
0.09
0.13
0.10
0.08
0.11
0.09
0.09
0.08
0.08
0.11
0.12
0.09
0.10
0.12
0.13
0.09
0.11
0.09
0.10
0.09
0.07
0.10
0.11
0.08
0.10
0.13
0.12
0.09
0.10
0.10
0.09
0.09
0.08
0.10
0.11
0.09
0.09
0.12
0.13
0.10
0.10
0.10
0.10
0.08
0.07
0.11
0.12
0.08
0.11
0.12
0.11
0.09
0.10
0.09
0.09
0.09
0.07
0.11
0.12
0.09
0.10
0.13
0.10
0.09
0.10
0.10
0.09
0.09
0.07
0.10
0.12
0.09
0.10
0.13
0.12
0.08
0.10
0.09
0.10
0.09
0.07
0.10
0.12
0.09
Imp-K
Imp-L
Imp-M
3. Materials and Methods
3.1. Chemicals and Reagents
Crude LDX and its impurities were synthesized in our laboratory. L-Lysine hydrochloride
), (S)-1-phenylethanamine ( ) and methanesulfonic acid were purchased from Energy Chemical
Corporation (Shanghai, China). The purity of all substances was >98%. HPLC-grade methanesulfonic
(2
3

Molecules 2018, 23, 3125
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acid were purchased from Fisher Scientific (Waltham, MA, USA). HPLC-grade acetonitrile (ACN) was
purchased from Honeywell (Newark, NJ, USA). Deionized water for preparing the aqueous phase was
obtained using a water purification system and all other chemicals were of analytical grade.
3.2. Analytical HPLC Conditions
Studies were conducted on a Dionex Ultimate 3000 HPLC instrument (Waltham, MA, USA)
equipped with a quaternary pump and a DAD detector. An analytical silica colum◦n YMC-Pack
ODS-AQ S5 120A (250
×
4.6 mm
×
5 µm, YMC, Nagoya, Japan) maintained at 30 C was used
for separation. Mobile phase A was 0.1% methanesulfonic acid (v/v) in water, while B was 0.1%
methanesulfonic acid in acetonitrile. The HPLC gradient program was set as follows: Time (min)/% of
solvent B: 0/3, 15/20, 30/50, 35/95, 37/95, 37.1/3, 45/3. The flow rate was 1.0 mL/min for a total run
time of 45 min, and the detection wavelength was 215 nm. The crude LDX was accurately weighed
and dissolved in the mixture of water and ACN (70:30, v/v) to obtain a test solution of 1.0 mg/mL.
Samples (10 µL) were injected into the HPLC system for analysis.
3.3. LC-MS Conditions
LC-MS was performed on an Agilent LC/MS system consisting of an Agilent 1260-LC system
equipped with a single quadruple mass detector and electrospray ionization (ESI) interface (Agilent
Technologies, Santa Clara, CA, USA). The column and mobile phase composition are the same as in
the HPLC analysis, except that formic acid was used instead of methanesulfonic acid. The LC gradient
program was set as follows: Time (min)/% of solvent B: 0/5, 40/15, 50/50, 55/95, 60/95, 60.1/5, 65/5.
The flow rate was 1.0 mL/min for a total run time of 65 min. The mass instrument was operated
in positive-ion ESI mode. Optimized mass conditions are as follows: drying gas (N2) flow rate of
◦
12.0 L/min, drying gas temperature 300 C, nebulizer pressure 50 psig, capillary voltage 3.0 kV. Scans
were acquired from 50 to 800 amu with a 0.1 s/scan. The high-resolution mass spectra and MS/MS
were recorded on a Q-TOF micro YA019 instrument (Waters, Milford, MA, USA).
3.4. NMR Spectroscopy
1H-NMR, 13C-NMR, distortionless enhancement by polarization transfer (DEPT), correlation
spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), and heteronuclear singular
quantum correlation (HSQC) NMR spectra were recorded on an Avance III 400 MHz spectrometer
(Bruker, Karlsruhe, Germany). Solvents used were DMSO-d6 or CDCl3.
3.5. FT-IR Spectroscopy
IR spectra were recorded in the solid state as KBr dispersions using a 670 FT-IR spectrophotometer
(NICOLET Waltham, MA, USA). Data were collected between 400 and 4000/cm, at a resolution of
4.0/cm.
3.6. Preparation of Standard and Sample Solutions
Samples was prepared using a water and acetonitrile mixture (70:30, v/v) as the diluent. In each
trial, the HPLC conditions were investigated by injecting test solution added with the twelve impurities
into the HPLC system. The concentration of LDX sample was 1.0 mg/mL, prepared by spiking the
twelve impurities (Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G, Imp-H, Imp-J, Imp-K, Imp-L
and Imp-M) into LDX at a concentration of 1.0 µg/mL and used to investigate the system suitability.
3.7. Forced Degradation Study
For forced degradation solutions, LDX was subjected to stress conditions according to ICH
guidelines [14]. The forced degradation of LDX was performed under hydrolytic (acidic and alkaline),
oxidative, thermal and photolytic conditions. The hydrolytic degradation was carried out separately

Molecules 2018, 23, 3125
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◦
in 1.0 M HCl (2.0 mL) as well as 5.0 M NaOH (2.0 mL) and kept in water bath at 90 C for 3 h.
The oxidative degradation was performed in 5% H2O2 (5.0 mL) at room temperature in the dark for 4 h.
◦
LDX was also subjected to thermolytic (90 C, 48 h) and photolytic (UV light, 4500 lx, 24 h) degradation.
After completion of the experiment, the samples were cooled to ambient temperature, neutralized with
a base or an acid, respectively. All of the stressed samples were kept at a concentration of 1.0 mg/mL
for assay determination.
4. Conclusions
An effective and selective HPLC method, used for the separation and determination of the
twelve impurities (process-related and degradation) in LDX bulk drug, was developed and optimized.
Structures of the two new compounds, Imp-H and Imp-M, were proposed by the synthesis route of
LDX and LC-MS analyses, and then confirmed and characterized using HRMS, ESI-MS/MS, 1D NMR
(1H-, 13C-, DEPT 135) and 2D NMR (COSY, HSQC, HMBC). Furthermore, probable mechanisms for
the formation of the process-related and degradation impurities were proposed based on the synthesis
route of LDX. The HPLC method was validated in terms of its linearity, accuracy, robustness, limits of
detection, and quantification. Full identification and characterization of these impurities is useful in
quality control in the manufacture of LDX.
Supplementary Materials: The following are available online, Table of contents; Figure S5 the LC-MS spectrum of
LDX and its impurities; Figure S6 The spectrum of Imp-B and Imp-C in normal-phase chromatography; Figure S7
NMR, HRMS and IR spectrogram of LDX and its impurities.
Author Contributions: S.G. designed and carried out the synthetic experiments, analyzed the data and wrote the
paper. L.M. performed HPLC analysis and other analysis work. C.Z. and F.Z. reviewed and edited the manuscript.
All authors read and approved the manuscript.
Acknowledgments: The authors are thankful to the teachers of the China State Institute of Pharmaceutical
Industry for supporting this study and the cooperation from other colleagues is also highly appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Biederman, J.; Krishnan, S.; Zhang, Y.X.; McGough, J.; Findling, R. Efficacy and tolerability of
lisdexamfetamine dimesylate(NRP-104) in children with attention-deficit/hyperactivity disorder: A phase
III, multicenter, randomized, double-blind, forced-dose, parallel-group study. Clin. Ther. 2007, 29, 450–463.
[CrossRef]
2.
3.
4.
5.
6.
Michel, T.; Krishnan, S.; Bishop, B.; Lauderback, C.; Moncrief, J.; Oberlender, R.; Piccariello, T. Abuse-resistant
Amphetamine Compounds. U.S. Patent 20050054561A1, 10 March 2005.
Michel, T.; Krishnan, S.; Bishop, B.; Lauderback, C.; Moncrief, J.; Oberlender, R.; Piccariello, T. Abuse-resistant
Amphetamine Prodrugs. U.S. Patent 20070042955A1, 22 January 2007.
Lu, C.Y.; Feng, C.H. Identification of dimer impurities in ampicillin and amoxicillin by capillary LC and
tandem mass spectrometry. J. Sep. Sci. 2007, 30, 329–332. [CrossRef] [PubMed]
Sun, Q.S.; Li, Y.; Qin, L. Isolation and identification of two unknown impurities from the raw material of
clindamycin hydrochloride. J. Sep. Sci. 2014, 37, 2682–2687. [CrossRef] [PubMed]
Carlos, G.; Comiran, E.; Herbstrith, M.; Limberger, R.; Bergold, A.; Froehlich, P. Development, validation and
comparison of two stability-indicating RP-LC methods using charged aerosol and UV detectors for analysis
of lisdexamfetamine dimesylate in capsules. Arab. J. Chem. 2016, 9, S1905–S1914. [CrossRef]
Guideline, I.H.T. ICH Harmonised Tripartite Guideline: Impurities in new drug substances Q3A(R2). In
Proceedings of the International Conference on Harmonization of Technical Requirements for Registration
of Pharmaceuticals for Human Use, Geneva, Switzerland, 25 October 2006.
7.
8.
9.
Yang, B.; Zhang, Y.J.; Zhang, S.S. Amidation of amines with eaters catalyzed by candida Antarctica lipase
(CAL). Cheminform 2010, 36, 1312–1316.
Routaboul, L.; Vanthuyne, N.; Gastaldi, S.; Gil, G.; Bertrand, M. Highly efficient photochemically induced
◦
thiyl radical-mediated racemization of aliphatic amines at 30 C. J. Org. Chem. 2008, 73, 364–368. [CrossRef]
[PubMed]

Molecules 2018, 23, 3125
16 of 16
10. Goudriaan, P.E.; Kaiser, J.; Ibrahim, H.; Verspui, G.A.; Cox, D.P. Process for the preparation of
lisdexamfetamine and related derivatives. U.S. Patent 20160376618A1, 29 December 2016.
11. Enders, D.; Haertwig, A.; Raabe, G.; Runsink, J. Diastereo- and enantio-selective synthesis of vicinal amino
alcohols by oxa-michael addition of N-formylnorephedrine to nitro alkenes. Eur. J. Org. Chem. 1998
1771–1792. [CrossRef]
,
12. Olah, G.A.; Fung, A.P.; Keumi, T. Oxyfunctionalization of hydrocarbons, hydroxylation of benzene and
alkylbenzenes with hydrogen peroxide in hydrogen fluoride/boron trifluoride. J. Org. Chem. 1981, 46,
4305–4306. [CrossRef]
13. Guideline, I.H.T. Validation of analytical procedures: text and methodology. In Proceedings of the
International Conference on Harmonization, Geneva, Switzerland, November 2005; pp. 11–12.
14. Guideline, I.H.T. Stability testing of new drug substances and products. Curr. Step 2003, 4, 1–24.
Sample Availability: Samples of the impurities A–M are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).