Journal of Pharmaceutical Sciences Acta Pharmaceutica Sinica 2014, 49 (1): 83 −88 · 83 ·

Study on the stability and pharmacokinetics of methylphenidate hydrochloride in plasma

Luo Xuemei 1, 2, Ding Li 2*, Gu Xin 1*, Jiang Liyuan 2, Dong Xin 2

(1. Department of Pharmacy, Drum Tower Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210008; 2. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, Jiangsu 21000

Summary: Study methylphenidate ( MPH) Instability in human plasma, established HPLC-MS/MS The determination method is also applied to the study of human pharmacokinetics.

use HPLC and LC-MS/MS The method was used to investigate the stability of methylphenidate in alcohol, water and plasma and the inhibitory effect of formic acid solution treatment on

methylphenidate degradation. the result shows, For freshly prepared human plasma samples ( 200 µL) Join now 2% Formic acid in water 10 µL, Can inhibit plasma esterase activity in plasma.

To 5 mmol·L −1 Ammonium acetate aqueous solution (containing 0.1% Formic acid) − Methanol ( 54 : 46) For mobile phase; use Sapphire C 18 Column. Using electrospray ion source ( ESI+) And

multiple response monitoring mode ( MRM) Carry out the detection, the detection ion is m/z 234.2 → 84.1 (MPH), m/z 260.3 →

183.1 ( Propranolol, Internal standard). The linear range of methylphenidate plasma concentration determination is 0.035 ~ 40 ng·mL −1; Intraday and intraday precision ( RSD) Are less than

5%, The accuracy is ± 2% within. Applied this method to study 6 Single dose oral methylphenidate in healthy volunteers 36 mg Post-pharmacokinetic characteristics. The established

method can accurately determine the plasma concentration of methylphenidate in human plasma, It can be used for human pharmacokinetic study of methylphenidate.

Key words: Methylphenidate; stability; pharmacokinetics; LC-MS/MS; hydrolysis

CLC number: R917; R969 Document identification code: A Article ID: 0513-4870 (2014) 01-0083-06

LC-MS/MS assay of methylphenidate: stability and

pharmacokinetics in human

LUO Xue-mei 1, 2, DING Li 2*, GU Xin 1*, JIANG Li-yuan 2, DONG Xin 2

( 1. Department of Medication, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China;
2. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China)

Abstract: The study aims to solve the instability problem of methylphenidate (MPH) in plasma, and establish a LC-MS/MS method for
simultaneous determining of MPH in human plasma. The stabilities of MPH in different media were studied, and the degradation characteristics
of MPH in these media were also investigated by HPLC and LC-MS/MS. To a 200 µL aliquot of freshly collected plasma sample, 10 µL 2%
formic acid was added immediately to prevent the hydrolysis of MPH in human plasma samples. Chromatographic separation was performed
on a Sapphire C 18 column using the mobile phase of methanol – 5 mmol·L −1 ammonium acetate buffer solution containing 0.1% formic acid (46 : 54).
MPH was quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. The
detection used the transitions of protonated molecules at m/z 234.2 → 84.1 for MPH and m/z 260.3 → 183.1 for propranolol (IS), separately. The
intra- and inter-assay precisions were all below 5.0%. The accuracies were all in standard ranges. The linear calibration curve was obtained in
the concentration range of 0.035−40 ng·mL −1. The methods fulfilled the demand. The method was used to determine the concentration of MPH
in human plasma after a single dose of 36 mg MPH tablet to 6 healthy Chinese volunteers. The method is suitable for the precisely
determination of MPH and for pharmacokinetic study of MPH in human plasma.

Key words: methylphenidate; stability; pharmacokinetics; LC-MS/MS; hydrolysis

Received date: 2013-07-04; Repair date: 2013-08-16.

* Corresponding Author Tel / Fax: 86-25-83271289, E-mail: dinglihg@sina.com

Tel / Fax: 86-25-83106666, E-mail: njglyygx@126.com
· 84 · Journal of Pharmaceutical Sciences Acta Pharmaceutica Sinica 2014, 49 (1): 83 −88

Methylphenidate hydrochloride ( methylphenidate, MPH), Commonly known as Ritalin ( ritalin), Collision energy 20 eV; Dry gas flow rate: 10 L·min −1; Spray chamber pressure: 40 psig; Drying gas

It is a synthetic central nervous system stimulant with a chemical structure similar to the temperature: 350 ℃. The selective detection of methylphenidate for mass-to-charge ratio is m/z 234.2

sympathetic amine drug amphetamine. Mainly used to treat children with attention deficit → 84.1, The internal standard (propranolol) is m/z

hyperactivity disorder ( attention-deficit hyperactivity disorder, ADHD)[ 1] . 260.3 → 183.1 .

Blood sample processing to 1.5 mL Plasma samples are precisely added to the

However, in human plasma, Methylphenidate hydrochloride is extremely unstable, and centrifuge tube 200 μL, 2% Concentrated formic acid in water 10 μL, Vortex to mix, then add

will be rapidly metabolized by plasma esterase to piperacinic acid ( ritalinic acid, RA)[ 2], internal standard (propranolol) reference solution ( 2.505 μg·mL −1)

It directly affects the accurate determination of methylphenidate concentration in human plasma. 20 μL, Vortex to mix. Add methanol 400 μL, Vortex 2 min, to

15 600 r·mL
In domestic and foreign literature, There are many pretreatment methods for the determination of methylphenidate −1 High2−14]:
in plasma[ speed centrifugation 5 min, Aspirate the supernatant 400 μL Transfer to the

Such as precipitation method [ 2] 、Liquid-liquid extraction ( LLE)[ 3−6] ,Solid Phase Extraction ( SPE)[ 7, 8] autosampler vial and inject 15 μL .

And derivation[ 9] law. but, The stability of methylphenidate in human plasma has not been Stability survey

reported in the literature. Some reports[ 4, 10] Mentioned fast at low temperatures ( 40 ~ 50 min) Preparation Under the same conditions, 3 The stability of different solvents at different storage time was

of methylphenidate plasma sample can slow down its rapid degradation in plasma. but, Because prepared with mass concentration of 10 μg·mL −1 Several samples of methylphenidate in methanol,

plasma esterase is at low temperature (such as ice bath, 0 ℃) Still active under conditions, Then water and plasma (at room temperature) , Placed at room temperature 0 , 1 , 2 , 4 ,

this method cannot completely inhibit the degradation of methylphenidate in plasma samples. 6 , 12 , 16 with 20 h Rear, Use high-performance liquid chromatograph for detection.

Not only that, in the repeated freeze-thaw process of the sample, Degradation will also occur[ 2] . Stable with or without formic acid treatment, same precipitation solvent, different storage

time The mass concentration of formulated methylphenidate is 10 μg·mL −1

Therefore, the above method is difficult to meet the high-throughput analysis often required in

pharmacokinetic research. This study uses a new pre-processing method, Very well inhibited the Of medicated plasma samples 2 Copies, and immediately to the sample 2 Add an appropriate

degradation of methylphenidate hydrochloride in human plasma, And established a tandem mass amount of formic acid (about 500 μL), Vortex to mix. Place two samples (sample 1 (Untreated

spectrometry analysis method. Established LC-MS/MS The analysis method is accurate, formic acid) Place at room temperature under the same conditions. in 0 , 1 , 2 , 4 ,

sensitive, convenient and durable, It can be used for human pharmacokinetic study of 6 , 12 , 16 with 20 h At the time point, after methanol precipitation HPLC Detection. At the same time,

methylphenidate hydrochloride. two groups 0 h The supernatant of the precipitated sample is 6 , 12 with

24 h Repeat the injections again.

Materials and Methods use LC-MS/MS Method to verify the preparation of several plasma samples containing

Instruments and reagents Agilent 1100 Liquid chromatograph, including dual methylphenidate, partly added 2% Formic acid 10 μL Vortex to mix. Place the plasma sample at

high-pressure pumps, autosampler, column thermostat, UV detector, chromatography workstation room temperature 12 h After use 2 Direct methanol precipitation, centrifuge to take supernatant LC-MS/MS

( Agilent Chemstation); Aglient 1200-6410B Liquid Chromatography − Mass spectrometer, Equipped analysis. in MRM In mode, monitor methylphenidate ( 234.2 → 84.1) With piperacetic acid ( 220.2 → 84.1)

with dual high-pressure pump, automatic sampler, electrospray ionization interface, triple .

quadrupole mass spectrometer detector and Agilent MassHunter Data processing software (USA Agilent square Legal investigation

Company); Reference product of methylphenidate hydrochloride was provided by a domestic Specificity 6 Blank plasma from different sources 200 μL,

pharmaceutical company (specifications: API) . In addition to not adding internal standards, Perform the rest according to the operation of "Blood Sample Processin

Solution preparation Precisely weigh methylphenidate hydrochloride 5.87 mg ( Equivalent LC-MS analysis, Obtained blank plasma sample chromatogram; methylphenidate and internal

to methylphenidate 5.08 mg) Put in 10 mL In a measuring flask, add methanol to dissolve and standard (propranolol) After the reference solution was added to the blank plasma, the same

dilute to the mark, shake well, and make up 508 μg·mL −1

method was used. Obtain the plasma sample chromatogram; take the plasma sample collected

Methylphenidate stock solution. Precisely weigh propranolol hydrochloride 5.71 mg ( Propranolol from healthy subjects after administration, and operate in the same way. A chromatogram of

equivalent 5.01 mg), Put in 10 mL In the measuring flask, add methanol to dissolve and dilute to plasma samples from healthy subjects was obtained.

the mark, shake well, Formulated into a mass concentration of 501 μg·mL −1 Propranolol stock Carry-over effect With the highest concentration point of the standard curve ( 40 ng·mL −1) Continuous

solution. Put in 4 Store at ℃. injection with a blank plasma sample 5 Group, to investigate the residual effect of the sample and

Chromatographic conditions Use chromatographic column Sapphire C 18 ( 150 mm × the internal standard ( carry-over effect). The criterion for the determination of residual effects is: In

2.1 mm ID, 5 μm, Sepax Tech.); Column temperature 38 ℃; mobile phase: blank plasma samples, the peak area of ​interference impurities at the sample retention time ( A s) Less

5 mmol·L −1 Ammonium acetate aqueous solution (containing 0.1% Formic acid) − Methanol ( 54 : than the lower limit of quantitation ( LLOQ) of

46), Flow rate 0.35 mL·min −1, Injection volume 15 μL . 20%, Interference impurity peak area at internal standard retention time ( A ′ s) Less than internal

Mass spectrometry conditions Using electrospray ionization source positive ion mode ( ESI+), standard 5% .

The ion detection method is multiple reaction monitoring ( MRM); Cracking voltage 95 V, Standard curve preparation and lower limit of quantification 1.5 mL Number of centrifuge tubes
 Luo Xuemei et al: Study on the stability and pharmacokinetics of methylphenidate hydrochloride in plasma · 85 ·

support, Accurately add different amounts of methylphenidate reference solution and blow dry with often. Two weeks before the experiment and during the experiment did not take any other drugs,

nitrogen flow, Add blank plasma 200 μL, Vortex and mix to prepare methylphenidate mass all signed an informed consent. Eat a unified diet during the trial. After the test subjects are

screened into the group according to the test protocol, they are given concentration ( Concerta ®
concentration as 0.035 56 , 0.101 6 , 0.304 8 ,

1.016 , 3.048 , 10.16 , 20.32 with 40.64 ng·mL −1 Of medicated plasma, Operate under the "Blood 36 mg/ sheet) One tablet for oral administration. On the day before taking the medicine twenty one : 00

Sample Processing" item, And prepare blank plasma samples at the same time, get on LC-MS/MS Start fasting later; the next morning 6 : 40 Collect a blank blood sample before administration, And

analysis, Record the chromatogram and prepare a standard curve. uniformly eat high-fat standard meals before administration, 7 : 00 Give one tablet (grain) under the

guidance of a doctor, use 240 mL Take the medicine with water and confirm with oral examination,

Precision and accuracy The mass concentration of prepared methylphenidate was 0.101 after administration 0.5 , 1 , 1.5 , 2 , 3 , 4 , 5 ,

6 , 2.032 with 35.56 ng·mL −1 Of medicated plasma, prepared for each concentration 5 Samples Press 6 , 8 , 10 , 12 , 16 , 20 , twenty four with 36 h Collect blood samples (total 15

the operation under "Blood Sample Processing" to calculate the ratio of the peak area of ​the Times 4 mL) . The collected blood sample is immediately transferred to the heparin

sample to the internal standard f . will f Substitute into the accompanying standard curve equation, Calculate
anticoagulation tube, shake well, 4 000 r·min −1 Centrifuge 5 min Take plasma, in duplicates, each 1

the concentration of each sample. Continuous measurement 3 Batch samples, according to mL, Join 50 µL of 2% Formic acid aqueous solution, vortex to mix,

sample concentration, Calculate accuracy and precision. −30 Store at ℃, For the determination of blood supply concentration.

Matrix effect, extraction recovery rate, precision and accuracy according to literature [ 15] Methods

to investigate matrix effects ( ME) And extraction recovery rate, the mass concentration of
Results and discussion
methylphenidate prepared and processed was 0.101 6 , 2.032 with 35.56 ng·mL −1 Of medicated
1 Stability inspection and selection of pretreatment methods
plasma, Each concentration is parallel 5 Copies, as a recovery sample; Take another 5 Blank
1.1 Under the same conditions, 3 Stability of different solvents at different storage times
plasma from different sources was treated in the same way, and the supernatant was taken, Add
the result shows: Methylphenidate placed in methanol 24 h No degradation (figure
methylphenidate and internal standard solution, Same as above 3 A mass concentration of
1, n = 3, RSD <5.1) . It degrades relatively slowly in water, 24 h Internal degradation 10% .
drug-containing plasma, Each concentration is parallel 5 Servings, As matrix effect sample and

recovery control sample; Use water instead of plasma to prepare and process the same method

as above 3 A mass concentration of solution, Each concentration is parallel 3 Parts, as a matrix

effect control sample. Inject the above sample, Record the chromatogram, and the ratio of the

peak area of ​the recovery rate and matrix effect sample to the average peak area of ​the

corresponding reference substance is the recovery rate and the matrix effect value. Then prepare

the above-mentioned equal concentration of drug-containing plasma, Prepare each concentration 5

Samples Operate under the "Blood Sample Processing" item, Calculate the ratio of sample and internal standard peak area f . will

f Substitute into the accompanying standard curve equation, Calculate the concentration of each

sample. Continuous measurement 3 Batch samples, According to the sample concentration, Calculate accuracy and precision.

stability The mass concentration of prepared methylphenidate was 0.101 6 ,

2.032 with 35.56 ng·mL −1 Of medicated plasma, Low Medium High 3 Several samples of each

concentration, Press “Operation of Blood Sample” to proceed immediately LC-MS/MS Analysis or

Figure 1 The degradation profiles of methylphenidate (MPH) at concentration level

placement in the injector 8.5 h Then test again; Take another 3 Served at room temperature 7.5
of 10 µg·mL −1 in methanol (M), water and plasma, separately, at room temperature
h, 3 Freeze-thaw 3 Times, then this 6 The blood samples were processed and determined in the

same way.

Pharmacokinetic studies Since the main target group of sustained release formulations 1.2 Stable with or without formic acid treatment, same precipitation solvent, different storage time The

of methylphenidate is children, Considering that children's medication may be affected by dietary result shows: sample 1 Still degraded quickly, 5 h degradation 30%, 24 h Internal degradation 50%

characteristics, This trial specifically investigates the pharmacokinetics of healthy subjects after ( Fig 2a, RSD <

high-fat standard meals. High-fat standard meal: It includes a creamy bread, an omelette, a piece 4.8%) . However, plasma treated with formic acid (sample 2), No obvious degradation was found

of cheese, a piece of bacon, a portion (Figure 2a) . Not only that, after methanol precipitation, the supernatant of the plasma sample

100 Gram fried potato cakes and 200 mL Whole milk. without formic acid treatment and the plasma sample after formic acid treatment (No obvious

6 Male healthy subjects, weight 50.0 ~ 70.5 kg, age degradation was found) Compared with it, there is still a weak degradation (Figure 2b, RSD < 4.0%),

This is related to water 20 h The degree of degradation is similar. at this time,

20 ~ 28 year old, Physical examination proves that liver and kidney functions are normal, and ECG is positive
· 86 · Journal of Pharmaceutical Sciences Acta Pharmaceutica Sinica 2014, 49 (1): 83 −88

Immediately add a certain amount of formic acid aqueous solution to the fresh plasma to ensure

the stability of methylphenidate. When measuring the blood concentration of methylphenidate,

attention should be paid to the handling and storage of plasma samples.

2 Methodological survey

2.1 Specificity From the picture 4 visible, Methylphenidate hydrochloride, internal standard peak

shape is good, Endogenous substances without interference, retention times are 2.0 with 4.3 min .

The method has high specificity, can accurately determine the concentration of methylphenidate

in plasma, and has high sensitivity.

2.2 Carry-over effect The results show that there is no peak of interfering impurities at the

retention time of the sample and the internal standard. There is no residual effect in this experiment.

2.3 Standard curve preparation and lower limit of quantitation The regression equation of methylphenidate:

f = 4.239×10 −3 x + 6.828×10 −2 ( r = 0.998 3), Weight coefficient

w = 1/ p 2, The minimum quantitation limit is 35.56 pg·mL −1, The accuracy is 100.5% ( n = 6), The

precision is 2.9% .

2.4 Matrix effect, extraction recovery, precision and accuracy test

Measured recovery of methylphenidate in plasma and ME See table 1, The recovery of internal

standard propranolol in plasma is ( 97.0 ± 3.7)% ( n = 15), The matrix effect is ( 105.2 ± 2.4)% ( n = 15),

The results showed that the recovery of methylphenidate and internal standard were good under

Figure 2 The degradation characteristics of MPH in human plasma (a) of the Set 1
(treated without acid) and the Set 2 (treated with acid). The residual amounts of
MPH (b) in the supernatant of the two sets in the following hours.
n = 3, x ± s
this experimental condition, and plasma matrix had no effect on its ionization and determination.
table 1 Indicate, intra-batch precision RSD Are less than 15%, Meet the requirements of biological
drug analysis and testing.

2.5 stability The results of the investigation of the stability of methylphenidate in plasma under

Plasma esterase should have been 2 The double volume of methanol precipitant destroys the different treatment conditions are shown in the table 2 . The results showed that adding one to fresh plasma

inactivation, and it is inferred that the added formic acid may weakly inhibit the chemical

equilibrium conversion between methylphenidate and acetic acid. Table 1 Precision, accuracy and recovery and matrix effects for the method. n = 5, x
±s
1.3 use LC-MS/MS Legal verification LC-MS/MS The analysis results of the processed samples are

shown in the figure 3 . The results confirm, In plasma samples of methylphenidate without formic RSD/% Recovery Matrix
C/ ng · mL −1 RE/%
/% effect/%
acid treatment, Methylphenidate is converted into piperacic acid in large quantities
Intra-day Inter-day

0.101 6 6.1 8.0 98.3 ± 4.2 107.3 ± 9.5 103.2 ± 3.1

( Retention time is 5.2 min), In the plasma sample of methylphenidate immediately after formic
2.032 4.1 5.8 99.8 ± 1.8 97.2 ± 3.5 96.7 ± 0.6
acid treatment, Piperacic acid has almost no peak.
35.56 4.8 6.1 100.7 ± 1.4 96.9 ± 3.0 97.4 ± 1.2
In summary, Methylphenidate degrades rapidly in plasma at room temperature, but

Figure 3 Stabilities of MPH in different conditions tested by LC-MS/MS drug-free plasma (a), MPH plasma samples without acid added (b), and plasma samples with acid
(c)
 Luo Xuemei et al: Study on the stability and pharmacokinetics of methylphenidate hydrochloride in plasma · 87 ·

Figure 4 MRM chromatograms for drug-free plasma (A), human plasma (200 μL) spiked with 10 µg·mL −1 of MPH and 100 ng·mL −1 IS (B), and plasma sample 3.0 h after oral
administration of 36 mg MPH (C)

Table 2 Stability of MPH in plasma at three QC levels. n=3

Freshly-prepared Short-term (7.5 h) Post-prepara tive (8.5 h) Freeze/thaw

Storage condition
Mean RE/% Mean RE/% Mean RE/% Mean RE/%

C/ ng · mL −1 0.101 6 0.103 6 2.0 0.096 87 −4.7 0.102 0 0.4 0.095 45 −6.1

2.032 1.981 −2.5 1.841 −9.4 1.846 −9.1 1.916 −5.7

35.56 36.71 3.2 34.07 −4.2 35.77 0.6 35.11 −1.3

Quantitative formic acid solution, Methylphenidate has good stability.

3 Pharmacokinetic parameters

Blood concentration − Time curve as shown 5 Shown, using DAS 2.0

The main pharmacokinetic parameters calculated by the data processing software are: methylphenidate

t 1/2 For ( 4.3 ± 1.0) h, AUC 0−36 h For ( 89.2 ± 12.8) ng∙h·mL −1,

AUC 0−∞ For ( 89.6 ± 12.9) ng∙h·mL −1, t max with C max Are

(6.8 ± 1.3) h with ( 8.3 ± 1.5) ng·mL −1; CL For ( 407.9 ± 48.5) L·h −1 . The pharmacokinetic parameters of

methylphenidate sustained-release tablets in this plasma are not significantly different from those

reported abroad.

Figure 5 Mean plasma concentration-time profile of MPH in six Chinese

in conclusion
volunteers after oral administration of a sustainedrelease (SR) tablet (36 mg/Tab,
Methylphenidate in plasma carboxylate hydrolase 1 (hCES1)[ 2] Under the effect of rapid Concerta®)
degradation, Converted to piperacetic acid. There is literature[ 16, 17]

Instability of methyl esters in plasma. When preparing the sample, the plasma sample is
Report, Formic acid can effectively inhibit carboxylate hydrolase in plasma 1 . The results of the

stability survey show that Adding formic acid can effectively inhibit the rapid degradation of precipitated with twice the volume of methanol, After centrifugation, the supernatant is taken and

methylphenidate in plasma, And to a certain extent, it inhibits the conversion of chemical the sample is injected LCMS/MS analysis. The sample analysis method is simple and easy to

equilibrium between methylphenidate and acetic acid. In addition, Because ① There may be operate, and the analysis time of each sample is controlled at 6 min, Greatly improve the efficiency

weak plasma esterase activity during the transportation, storage and repeated freezing and of determination, suitable for high-throughput analysis.

thawing of untreated plasma samples; ②Adding formic acid directly to whole blood may cause The pharmacokinetic study uses a high-fat diet after meals, because the main target

blood cells to rupture, Hemolysis occurs, The determination of methylphenidate in plasma group of drugs is children, Children exposed to high-fat diet at breakfast (Such as eggs, milk, etc.) It

samples is affected. Therefore, finally take formic acid as soon as whole blood is made into is more likely and more common than the general population. Existing literature[ 18, 19] Reports on

plasma, Then immediately freeze the plasma sample, Until analysis and determination. studies between methylphenidate and high-fat diets, although the results of the study show that

high-fat diets have little effect on pharmacokinetics of methylphenidate in vivo, but

This study uses the addition of freshly prepared plasma samples 5% comprehensive consideration, The experimental plan was finally selected as a unified high-fat

Volumetric 2% The pretreatment method of formic acid aqueous solution, Better inhibition of piper diet.
· 88 · Journal of Pharmaceutical Sciences Acta Pharmaceutica Sinica 2014, 49 (1): 83 −88

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