±threo-Ritalinic acid-D10 Hydrochloride an internal standard for quantitation of Ritalinic acid by LCMSMS: Synthesis determination of isotopic distribution by qNMR and LCMS

By: Elizabeth B. Marek, Joshua Cooper, Huahua Jian, Uma Sreenivasan, Isil Dilek,

Abstract

As a major metabolite of methylphenidate (1, Ritalin), ±-threo-Ritalinic acid2 is of clinical relevance. To this end ±-threo-ritalinic acid-D10HCl3 was synthesized in seven steps with a purity of 99% and an isotopic purity ratio of D0/D10= 0% and a significant amount of the D9-D7 isomers. Because practical ion monitoring is based on the ratio of D0/D10, the standard was found to be suitable for use as an internal standard in LC-MS/MS analysis of ritalinic acid and related compounds. The presence of significant amounts of the D9 isomer prompted extensive structure elucidation work using LC-MS/MS scrambling and 1D, 2D, and qNMR techniques.

Introduction

Methylphenidate, most commonly known by the Novartis trade name Ritalin®, is a pyschostimulantused to treat attention-deficit hyperactivity disorder, postural orthostatic tachycardia syndrome, and narcolepsy, by increasing alertness and attention and by counteracting fatigue. Methylphenidate was originally sold as a mixture of diastereomers, although it has been shown that the majority of the activity is attributed to the ±-threo isomer. More recent products such as Focalin® contain only the active ±-threo isomer. While analytical reference standards of the diastereomericmixture of methylphenidate and its metabolites are available, standards containing only the active isomer are now desirable to reflect the current directive of using only the active isomer in drug products. Therefore it is also desirable to synthesize stable-labeled derivatives of the active isomers of methylphenidate and its metabolites, such as ritalinicacid, for use as internal standards.

Ritalinicacid is a major metabolite of and synthetic precursor to methylphenidate and may be monitored clinically and forensically. The synthesis of deuterated±-threo-ritalinic acid was therefore undertaken to develop an analytical reference standard and as a precursor to deuterated ±-threo-methylphenidate.

Synthesis of ±-threo-Ritalinic acid-D10HCl

Based on literature precedence 1-3, ±-threo-Ritalinic acid-D10 HCl was synthesized in seven steps from pyridine-D5. During the synthesis, the crucial reduction of the pyridine moiety to the fully deuterated piperidine proceeded in good yield but LC/MS-SIM indicated that the product contained a mixture of 55% D10, 34% D9and 11% D8-D7. The presence of the D0 isomer was not detected. This deuterium ratio was carried through to the final product.

Synthesis of ±-threo-Ritalinic acid-D10 HCl

References

  1. Heterocyclic Chem. 44; 2007; 1485.
  2. US Patent 5936091.
  3. J. Med. Chem. 39; 6; 1996; 1201.

 

Characterization of ±-threo-Ritalinic acid-D10HCl

 

Isotopic distribution by LC/MS-SIM
Deuterium Content Mole Percent
(xi)
D10 55.16%
D9 33.98%
D8 9.21%
D7 1.48%
D6 0.148%
D5 0.0128%
D4-D0 0.0%

 

The identity of ±-threo-Ritalinic acid-D10 HClwas established through NMR and mass spectrometry. The chemical purity was established through HPLC/UV, Karl Fisher, GC/FID Headspace and ROI. LC-MS/MS studies were performed to evaluate isotopic purity, deuterium distribution, fragmentation patterns and suitability for use as an internal standard. HPLC analysis indicated a purity of 99% with isotopic purity ratio D0/D10 = 0% by LC/MS-SIM. Additionally, LC/MS-SIM confirmed the presence of 45% D9-D7 isomers (see isotopic distribution at left).

Isotopic distribution by quantitative NMR

Using maleic acid as an internal standard, Quantitative NMR (qNMR) was used to determine the percentage of hydrogen and therefore deuterium on each carbon of ±-threo-Ritalinic acid-D10 HCl.

Isotopic distribution by quantitative NMR

Isotopic distribution by quantitative NMR

H% = % hydrogen
I = integral of signal
n = number of protons under the signal of interest
m = mass of compound of interest
M = molecular weight
std = internal standard
A or a = component or analytewhose purity is to be calculated
P = purity

qNMR confirmed the presence of a nearly even distribution

qNMR confirmed the presence of a nearly even distribution of deuterium throughout the piperidinemoiety and also revealed that a small percentage of the hydrogenson the aromatic moiety had exchanged for deuterium as well. These results suggest that the Pt/C catalyst used in step 5 of the synthesis also facilitated the exchange of aromatic hydrogensfor deuterium.

 

Product Internal Standard
±-threo-Ritalinic acid-D10 Maleic acid
Mass balance Istd = 2000 Ma = 265.223
Purity Pa = 95.51% Mstd = 116.07
  mstd = 4.029 ma = 15.552
  nstd = 2 Pstd = 99.78%
Position nA IA H% D%
Ortho 2 3122.0571 96.540 3.460
Para&meta 3 4708.465 97.063 2.937
Aromatic 5 7830.522 96.854 3.146
2 1 61.743 3.818 96.182
3 1 130.420 8.066 91.934
4 2 158.628 4.905 95.095
5 2 264.270 8.172 91.828
6 2 185.778 5.745 94.255
7 2 208.357 6.443 93.557

 

The molar mass of the product is calculated based on isotopic purity results obtained by LC/MS-SIM.

Investigation of ±-threo-Ritalinic acid-D10 HCl Scrambling using Waters Xevo G2 Q-Tof

Investigation of ±-threo-Ritalinic acid-D10 HCl Scrambling using Waters Xevo G2 Q-Tof

Effect of Resolution on the Scrambling of ±-threo-Ritalinic acid-D10 HCl

Waters XevoG2 Q-Tof

Waters XevoG2 Q-Tof

Agilent 6410 triple quadrupole

Compound MS1 Resolution Collision Energy Label Transition(s) dn Scrambling %
dn-1/ dn
Ritalinic Acid Unit 20 d10 230.2→93.2 11.19
native 220.1→84.1 0.45
10 d10 230.2→230.2 0.39
native 220.1→220.1 0.3
Wide 20 d10 230.2→93.2 11.31
native 220.1→84.1 0.46
10 d10 230.2→230.2 0.4
native 220.1→220.1 0.31
Widest 20 d10 230.2→93.2 51.46
native 220.1→84.1 0.46
10 d10 230.2→230.2 47.29
native 220.1→220.1 0.31

 

Scrambling was seen on both the Waters XevoG2 Q-Tofand the Agilent 6410 triple quadrupole, although both instruments indicated the importance of higher resolution. By increasing the resolution, the scrambling was mitigated.

Investigation of the use of ±-threo-Ritalinic acid-D10 HCl as an Internal Standard

Agilent UHPLC 1290 HPLC-6460 triple quad ESI+
0.4 mL/min
5 μL injection volume
85:15 A:B
A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Internal Standard: 500 ng/mLin methanol

 

Compound Fragmentation (V) CE (V) Transition(s) dn
-threo-Ritalinic acid-D10 HCl 102 20 230.2→93.1
Ritalinic Acid HCl 102 20 220.14→84.1

 

Concentration linear range: 1-10,000 ng/mLin methanol

Concentration linear range: 1-10,000 ng/mLin methanol

Conclusions

  • ±-threo-Ritalinic acid-D10 HCl was synthesized in good yield, sufficient ratio of D0/D10, and 99% purity.
  • LC-MS/SIM indicated significant amounts of D9-D7 with no D0.
  • The percentage of deuterium present on each carbon was determined by qNMR.
  • LC-MS/MS studies indicated that ±-threo-Ritalinic acid-D10 HCl fragments well and that deuterium scrambling can be minimized.
  • Standard curve supports the use of ±-threo-Ritalinic acid-D10 HCl as an effective internal standard.

Materials

     
Related Links