pharmaceutics

Article HPLC-Based Analysis of Impurities in Sapropterin Branded and Generic Tablets

Emanuela Scudellaro, Luciana Tartaglione, Fabio Varriale, Carmela Dell’Aversano and Orazio Taglialatela-Scafati *

Department of Pharmacy, School of Medicine and Surgery, Universita’ degli Studi di Napoli Federico II, Via D. Montesano, 49, 80131 Naples, Italy; [email protected] (E.S.); [email protected] (L.T.); [email protected] (F.V.); [email protected] (C.D.) * Correspondence: [email protected]; Tel.: +39-081-678-509

 Received: 3 February 2020; Accepted: 31 March 2020; Published: 3 April 2020 

Abstract: This work was aimed at the definition of a chromatographic method able to separate and quantify impurities present in sapropterin-containing drugs during an accelerated stability study. The chromatographic method was applied to the orphan drug Kuvan® and to its corresponding generic sapropterin Dipharma (Diterin®), both of which are approved for the treatment of hyperphenylalaninemia-induced symptoms. The two products tested had a similar manufacture date and both had an approved stability shelf-life of three years. Samples were analyzed by HPLC at T = 0 and after six months of storage at 40 ◦C and 75% relative humidity. Identification of the impurities was supported by a detailed mass spectrometry and MS/MS profile. The analysis demonstrated an overall higher stability for the Diterin® formulation, which was related to a lower increase of some impurities compared to Kuvan®.

Keywords: sapropterin; PKU; BH4 deficiency; chemical content; impurity identification; HPLC-UV; MS/MS

1. Introduction Sapropterin dihydrochloride, (6R)-L-erythro-5,6,7,8- dihydrochloride (BH 2HCl), referred also as sapropterin, is a synthetic version of the naturally occurring 6R-BH , 4· 4 which is, among others, a cofactor of -, tyrosine-, and tryptophan hydroxylases. Sapropterin is the active ingredient of the drug Kuvan®, a medicine approved by the Food and Drug Administration (FDA) in 2007 and the European Medicines Agency (EMA) in 2008, for the treatment of hyperphenylalaninaemia (HPA), a rare disease caused by defects in the phenylalanine hydroxylase (PAH) [1] or by a defect in the biosynthesis or recycling of BH4. Mutations in the phenylalanine hydroxylase gene (PAH) lead to (PKU) which, depending on the seriousness of the mutations, can be mild (Phe levels 300–600 µM), moderate (Phe levels 600–1200 µM), or severe (Phe levels >1200 µM). On another side, defects in BH4 synthesis or recycling lead to BH4 deficiency. The latter occurs in only around 2% of the HPA patient population [2]. In Europe, patients who are found to respond to a seven-day BH4 loading test (30% decrease of phenylalanine level in the bloodstream), could benefit from a life-long treatment with Kuvan®, at a daily dose of between 5 and 20 mg/kg [3].

Pharmaceutics 2020, 12, 323; doi:10.3390/pharmaceutics12040323 www.mdpi.com/journal/pharmaceutics

Article HPLC-Based Analysis of Impurities in Sapropterin Branded and Generic Tablets

Emanuela Scudellaro 1, Luciana Tartaglione 1, Fabio Varriale 1, Carmela Dell’Aversano 1 and Orazio Taglialatela-Scafati 1,* Department of Pharmacy, School of Medicine and Surgery, Universita' degli Studi di Napoli Federico II, Via D. Montesano, 49, 80131 Naples, Italy; [email protected] (E.S.); [email protected] (L.T.); [email protected] (F.V.); [email protected] (C.D.) * Correspondence: [email protected] Tel.: 0039-081-678509

Received: 3 February 2020; Accepted: 31 March 2020; Published: date

Abstract: This work was aimed at the definition of a chromatographic method able to separate and quantify impurities present in sapropterin-containing drugs during an accelerated stability study. The chromatographic method was applied to the orphan drug Kuvan® and to its corresponding generic sapropterin Dipharma (Diterin®), both of which are approved for the treatment of hyperphenylalaninemia-induced symptoms. The two products tested had a similar manufacture date and both had an approved stability shelf-life of three years. Samples were analyzed by HPLC at T = 0 and after six months of storage at 40 °C and 75% relative humidity. Identification of the impurities was supported by a detailed mass spectrometry and MS/MS profile. The analysis demonstrated an overall higher stability for the Diterin® formulation, which was related to a lower increase of some impurities compared to Kuvan®.

Keywords: sapropterin; PKU; BH4 deficiency; chemical content; impurity identification; HPLC-UV; MS/MS

1. Introduction Sapropterin dihydrochloride, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin dihydrochloride (BH4·2HCl), referred also as sapropterin, is a synthetic version of the naturally occurring 6R-BH4, which is, among others, a cofactor of phenylalanine-, tyrosine-, and tryptophan hydroxylases. Sapropterin is the active ingredient of the drug Kuvan®, a medicine approved by the Food and Drug Administration (FDA) in 2007 and the European Medicines Agency (EMA) in 2008, for the treatment of hyperphenylalaninaemia (HPA), a rare disease caused by defects in the phenylalanine hydroxylase (PAH) [1] or by a defect in the biosynthesis or recycling of BH4. Mutations in the phenylalanine hydroxylase gene (PAH) lead to phenylketonuria (PKU) which, depending on the seriousness of the mutations, can be mild (Phe levels 300–600 µM), moderate (Phe levels 600–1200 µM), or severe (Phe levels >1200 µM). On another side, defects in BH4 synthesis or recycling lead to BH4 deficiency. The latter occurs in only around 2% of the HPA patient population [2]. In Europe, patients who are found Pharmaceutics 2020, 12, 323 2 of 10 to respond to a seven-day BH4 loading test (30% decrease of phenylalanine level in the bloodstream), could benefit from a life-long treatment with Kuvan®, at a daily dose of between 5 and 20 mg/kg [3]. O OH H N 6 N OH H2N N N H H

Sapropterin

PharmaceuticsSapropterin 2020, 12,is x; adoi: relatively complex and unstable molecule, whosewww.mdpi.com/journal/ synthesis is madepharmaceutics difficult by the presence of three adjacent asymmetric centers. Until 1999, sapropterin was obtained as a mixture of 6R/6S diastereomers in a 69:31 ratio. However, only the 6R isomer is biologically active, the 6S isomer has been reported to cause an irreversible inactivation of rat liver PAH [4]. HPA commonly requires life-long treatment; therefore, the medicine must be of the highest quality and stability in order to avoid the ingestion and accumulation of potentially dangerous impurities over the patient’s life. Over the years, some papers have raised doubts about the quality of some generic drugs, which are indicated to be of lower quality compared to their branded counterparts [5]. Such papers prompted FDA Commissioner, Dr Scott Gottlieb, and the Center for Drug Evaluation and Research (CDER) Director, Dr Janet Woodcock, to publish a statement to deny any alleged lower safety and/or effectiveness of FDA approved generics versus their corresponding branded drugs [6]. Notwithstanding this initiative, the feeling that branded drugs cannot indiscriminately be exchanged by their corresponding generics, is widely spread among practitioners and some patient communities. The aim of the present study was to experimentally verify this perception by investigating the composition and stability of the life-long treatment sapropterin-containing drug Kuvan® and its generic sapropterin Dipharma (Diterin®). Furthermore, our data, based on HPLC-UV and LC-high resolution mass spectrometry (LC-HRMS) analysis, moved a step forward in providing the most detailed profile available to date of the impurities present in sapropterin-containing products, leading to the identification of nine by-products.

2. Materials and Methods

2.1. Material Kuvan®, batch number L141191, was purchased in an ordinary manner from a pharmacy with expiry date: 02/2020. The manufacture date was not available; however, the leaflet of Kuvan® indicated a 3-year shelf-life indicating a manufacture date of around February 2017. Diterin® was generously donated by Dipharma SA (Chiasso, Switzerland), batch No. 17107H2, manufacture date: 05/2017, and expiry date: 04/2020. Starting from T = 0, both products were stored at 40 ◦C and 75% Relative Humidity (RH) in a constant climate chamber model HPP110 (Memmert, Italy), V = 108 L. Samples of both products were -taken at T = 0 and 6 months for the purpose of analysis. Storage at 40 ◦C and 75% RH for 6 months are the conditions indicated by EMA for accelerated stability studies (Note for Guidance on Stability Testing, CPMP/ICH/2736/99). Water HPLC Plus, acetonitrile for HPLC ( 99.9%), ammonium formate (reagent grade, 97%), ≥ formic acid (reagent grade, 95%), and sapropterin, were all purchased from Sigma Aldrich (Milan, ≥ Italy). Sodium dihydrogen phosphate dihydrate (NaH PO 2H O), phosphoric acid 85% (H PO ), and 2 4· 2 3 4 ascorbic acid were purchased from Titolchimica S.p.A (Pontecchio Polesine, Italy). HPLC-UV analysis was carried out on a Shimadzu LC-10AD instrument equipped with detector, Shimadzu SPD-10A (Shimadzu Italia, Milan, Italy). A clarity software program was used for the chromatogram analysis. Pharmaceutics 2020, 12, 323 3 of 10

2.2. Methods

2.2.1. HPLC Kuvan® and Diterin® tablets were pulverized, after which 7.0 mg of the obtained fine powders were accurately weighed, transferred into test tubes, and dissolved in 7.0 mL of water containing 0.2% ascorbic acid (w/v). Given the reported solubility of sapropterin in water (2.0 mg/mL), we assumed that it was fully solubilized in these conditions; however, in order to eliminate insoluble excipients, the solution was sonicated in an ice bath for 15 min with intermittent shaking, and then centrifuged at 3000 rpm (1.21 g) for 20 min (MPW-56 centrifuge, MPW instruments, Warszawa, Poland). The clear × supernatant was collected and 20 µL was immediately (roughly 40 min after tablet pulverization) analyzed by HPLC-UV. Chromatographic separation was performed in the isocratic mode, using an ion-exchange Partisil® column 10 SCX-250A, 250 4.6 mm i.d, 5 µm particle size. Mobile phase was × 0.03 M NaH2PO4 solution—pH adjusted to 3.0 with phosphoric acid (H3PO4). The flow rate was set at 1.2 mL/min and the run time was 20 min. The injection volume was 20 µL and the UV detector was set at 265 nm. The percentage concentration of the impurities was calculated as follows:

% Concentration = (Ap / At) 100 × where Ap = area of the impurity peak, At = total area of the impurities and sapropterin. Three independent measurements were carried out for each sample.

2.2.2. Mass Spectrometry LC-HRMS experiments were carried out on a Dionex Ultimate 3000 system, which included a solvent reservoir, in-line degasser, a quaternary pump and refrigerated autosampler, and column oven coupled to a hybrid linear ion trap LTQ Orbitrap XLTM Fourier Transform MS (FTMS) equipped with an Electrospray (ESI) ION MAXTM source (Thermo-Fisher, San Josè, CA, USA). Separations were performed on a 150 mm 2.0 mm ID column packed with 3 µm TSK-GEL® Amide-80 material (Sigma × Aldrich) at room temperature. Mobile phases were (A) water and (B) 95% acetonitrile and 5% water (v/v), both containing 2.0 mM ammonium formate and 3.6 mM formic acid (pH 3.55). A gradient elution, setting both flow rate and injection volume at 0.2 mL/min and 5 µL respectively, was run starting from 90% B (t = 0) and held for 20 min (t = 20), it was then decreased from 90% to 20% B in 1 minute (t = 21) and held for 10 min (t = 31), it was then increased to 90% B in 1 minute (t = 32) and held for 10 min (t = 42) to re-equilibrate the column. A standard solution of 6R-(BH4) at 10 µg/mL (ascorbic acid 0.2% w/v) was prepared, and used to set up the source parameters while operating in a positive ionization mode, as follows: capillary temperature = 360 ◦C, sheath = 46 and auxiliary gas = 7.5 (arbitrary units) spry voltage = 4.8 kV, capillary voltage = 24 V, and tube lens = 70 V. Full scan spectra were recorded in the range m/z 150–300. HR collision-induced dissociation (CID) experiments were carried out selecting the [M+H]+ ion of each compound as precursor and collision energies (CEs) (depending on the precursor ion), in the range of 22% to 25%, isolation width = 2.0, activation Q = 0.250, and activation time = 30 ms. In all the experiments, the resolving power (RP) was set at 60,000 (FWHM at m/z 400). Elemental formulae were calculated using the mono-isotopic ion peak of each cluster through Thermo Xcalibur software v2.2 SP1.48 (Thermo Fisher, San Josè, CA, USA) at a 5 ppm mass tolerance. + A full scan HRMS spectrum of BH4 standard solution, showed the characteristic and dominant [M+H] ion at m/z 242.1248 (C9H16N5O3, Ring Double Bond Equivalent RDB = 4.5, ∆ppm = 0.637), and the + relevant [M+Na] adduct ion at m/z 264.1069 (C9H15N5O3, RDB = 4.5, ∆ppm = 0.717) with a relative abundance of 80–90% for the pseudomolecular ion. A mixture containing each standard at 10 µg/mL (in water containing ascorbic acid 0.2% w/v) was prepared and injected under the same experimental conditions. Then, the LC-HRMS spectrum was acquired, and through its interpretation, as reported for + BH4, the presence of the peculiar [M+Na] ion was highlighted. However, its relative abundance, with respect to the relevant [M+H]+ ion, was characteristic for each compound. Pharmaceutics 2020, 12, 323 4 of 10

3. Results

3.1. Individual Impurity Identification Finely pulverized, Kuvan® and/or Diterin® tablets were dissolved in 7.0 mL of a 0.2% (w/v) solution of ascorbic acid in water, sonicated, centrifuged and analyzed by HPLC-UV in isocratic mode on an ion-exchange Partisil® column 10 SCX-250A, 250 4.6 mm i.d, 5 µm, using 0.03 M × NaH2PO4 water solution, with pH adjusted to 3.0. Peaks were detected with the UV detector set at λ 265 nm. This analytical method was selected on the basis of its capability to separate and detect nine impurities/degradation products, providing better results compared to other methods present in Pharmaceutics 2020, 12, x 4 of 10 the literature [7,8]. Figure1 reports the HPLC profile obtained for Kuvan ® tablet at T = 0. The two majorpeaks peaks were were easily easily identified identified as ascorbic as ascorbic acid (RT= acid (RT2.94 =min)2.94 and min) sapropterin and sapropterin (RT= 8.61 (RT min)=, 8.61and min), and confirmedconfirmed by by injection injection of ofstandards. standards.

Ascorbic Acid Sapropterin

1 2 3 4 6 7 5 8 9

® FigureFigure 1. HPLC 1. HPLC profile profile of of pulverized pulverized KuvanKuvan® tablettablet at atT T= 0= dissolved0 dissolved in 7.0 in mL 7.0 of mL a 0.2% of a 0.2%(w/v) (w/v) solutionsolution of ascorbic of ascorbic acid acid in in water. water. HPLC-UV HPLC-UV (λ 265 nm) nm) was was run run in inisocratic isocratic mode mode on an on ion an-exchange ion-exchange ® ® PartisilPartisilcolumn column using using 0.03 0.03 M M NaH NaH22PO44 waterwater solution solution (pH (pH 3.0) 3.0) as an as eluent. an eluent.

NineNine minor minor compounds compounds ( 1(1––99)) attributable to to impurities/degradation impurities/degradation products products were detected were detected at at retentionretention timestimes rangingranging from RT RT == 3.683.68 to to 15.1 15.1 min. min. The The origin origin of these of these compounds compounds could could be be attributableattributable either either to smallto small impurities impurities in in thethe sapropterin,sapropterin, deriv derivinging from from its itssynthetic synthetic procedure procedure,, or to or to products of its degradation. During the last few decades, sapropterin has been the subject of intense products of its degradation. During the last few decades, sapropterin has been the subject of intense investigation aimed at identifying its degradation products, which mainly originate from oxidation investigation aimed at identifying its degradation products, which mainly originate from oxidation and hydrolysis [7,8], and these products are the obvious candidates for the assignment of the and hydrolysisstructures of [7 compounds,8], and these 1–9 products. Since the are majority the obvious of them candidates were commercially for the assignment available, ofinjection the structures of of compoundsthe standards1–9 .in Since the thesame majority HPLC ofconditions them were, allowed commercially for the available,confident injectionidentification of the of standards the in thecompounds same HPLC as conditions,reported in allowed Table for1. theIn confidentparticular, identificationcompound 6 of was the compoundsassigned as as(6 reportedR)- in Tabletetrahydrobiolumazine1. In particular, compound, since it co6-elutedwas assigned with the ascommercially (6R)-tetrahydrobiolumazine, available product of since sapropterin it co-eluted withhydrolysis, the commercially while the available isomeric product compound of sapropterin 7, was assigned hydrolysis, as (6S while)-tetrahydrobiolumazine the isomeric compound by 7, was assignedcomparison as with (6S)-tetrahydrobiolumazine the synthetic biolumazine bymixture comparison, which was with obtained the synthetic by following biolumazine procedures mixture, whichfrom was the obtained literature by following[9,10]. Similarly, procedures compound from the3 was literature assigned [9, 10as]. 7,8 Similarly,- compound by3 was comparison with the commercially available standard, while the isomeric compound 4 was assigned as 7,8-dihydrobiopterin by comparison with the commercially available standard, while the tentatively assigned as 5,6-dihydrobiopterin. The assignment of compounds 1–9 was supported by a isomeric compound 4 was tentatively assigned as 5,6-dihydrobiopterin. The assignment of compounds detailed LC-HRMS and (MS/MS) analysis carried out on standards and on the tablet (see Section 3.2). 1–9 was supported by a detailed LC-HRMS and (MS/MS) analysis carried out on standards and on the tablet3.2. (see Mass Section Spectrometry 3.2). Analysis Although a few liquid chromatography-tandem mass spectrometry methods (LC-MS/MS) are available to determine tetrahydrobiopterin [11–13], there is shortage of spectrometric data acquired on high resolution MS systems. Therefore, LC-HRMSn (n = 1,2) spectra of each available impurity (1– 3 and 5–9) were acquired and analyzed in order to investigate the fragmentation pattern. Mobile phase selection was made taking into account the pH used for the above described LC-UV detection (pH = 3.5). Considering that the use of a phosphate buffer is not compatible with ESI-MS ionization and that the analyzed compounds are small-size highly polar molecules, hydrophilic interaction liquid chromatography (HILIC) was selected to chromatographically resolve these compounds. Each standard was separately injected onto the column at a concentration level of 10 µg/mL and the full HRMS spectra obtained, were in all cases dominated by the [M+H]+ ion with the presence of a minor sodium adduct (Table S1, Supporting Information). Each standard was analyzed within 90 min from sample preparation in order to minimize degradation phenomenon that would result in the formation of oxidation products and a consequent dramatic signal decrease. Figure 2 reports Extracted Ion Chromatograms (XICs) of the standards, obtained by selecting the relevant exact masses of each compound. Collision induced dissociation (CID) HR-MS2 spectra associated with each peak were also acquired (Figure S1, Supporting Information), and the elemental compositions

Pharmaceutics 2020, 12, x 5 of 10

PharmaceuticsPharmaceutics 20 202020,, 1, 21,,2 xx, x 5 5of of 10 10 Pharmaceutics 2020, 12, x ®5 of 10 forPharmaceutics all the obtained2020, 12, x fragment ions are reported in Table S2 (Supporting Information). Kuvan 5 ofand 10 ® Diterin tablets were then dissolved using the procedure reported in the Materials and Methods®® forPharmaceuticsfor all all the the obtained20 obtained20, 12, x fragment fragment ions ions are are reported reported in in Table Table S 2S 2(Supporting (Supporting Information). Information). Kuvan Kuvan®5 andof and 10 ® ® sectionDiterin® (2.2.2) tablets and were analyzed then versusdissolved the standardsusing the underprocedure the same reported experimental in the Materials conditions. and Methods DiterinPharmaceuticsfor all the® tablets obtained2020, 1were2, x fragment then dissolved ions are using reported the procedurein Table S 2reported (Supporting in the Information). Materials and Kuvan Methods5 ofand 10 section® (2.2.2) and analyzed versus the standards under the same experimental conditions. ® sectionPharmaceuticsforDiterin all the (2.2.2) tablets obtained2020 and, 1were2, xanalyzed fragment then dissolvedversus ions arethe using standardsreported the inprocedure under Table the S2 samereported (Supporting experimental in the Information). Materials conditions. and Kuvan Methods5 ofand 10 Table 1. Assignment of minor compounds detected for Kuvan® tablet at T = 0. forDiterinsection all the ®(2.2.2) tablets obtained and were analyzed fragment then dissolved versus ions arethe using reportedstandards the inprocedure under Table the S2 reported same(Supporting experimental in the Information). Materials conditions. and Kuvan Methods® and ® Table 1. Assignment of minor compounds detected for Kuvan® tablet at T = 0. DiterinsectionforPeak all the ®(2.2.2) tablets obtained andTable were analyzed fragment1. then Assignment dissolved versus ions of arethe minor using standardsreported compounds the procedurein underChemical Table detected the S2 reportedsame (Supporting for Kuvan experimental Standardin® thetablet Information). Materials at RTconditions. T = 0. and KuvanSample Methods® and RT ® ® Table 1. AssignmentName of minor compounds detected for Kuvan tablet at T = 0. NsectionDiterinumberPeak (2.2.2) tablets and were analyzed then dissolvedversus the using standards the procedure underSChemicaltructure the samereported experimental inStandard the(min) Materials conditions. RT and SampleMethods(min) RT Peak Chemical Standard® RT Sample RT section (2.2.2) andTable analyzed 1. AssignmentNameName versus of the minor standards compounds under detected the same for Kuvanexperimental tablet at conditions. T = 0. PharmaceuticsNNPumberumbereak 2020 , 12, 323 Name ChemicalSStructuretructure Standard(min)(min) RT Sample(min)(min) RT 5 of 10 Number Table 1. AssignmentName of minor compoundsStructureO detectedO H for Kuvan® tablet(min) at T = 0. (min) NPumbereak ChemicalStructureN Standard(min) RT Sample(min) RT N O OH ® 1 Table 1. AssignmentBiopterinName of minor compoundsO detectedOH for Kuvan tablet3.65 at T = 0. 3.68 O OHOH NPumbereak ChemicalStructureNN Standard(min) RT Sample(min) RT H2N NON N OH ® 1 Table 1. AssignmentBiopterinName of minor compoundsN H detected for Kuvan3.65tablet at T = 0. 3.68 NPumbereak1 ChemicalStructureN OH Standard(min)3.65 RT Sample(min)3.68 RT H NN N N OH Name H2N2 NO N OH 1 Biopterin H2N NHH N 3.65 3.68 Number StructureH N OH (min) (min) Peak Number Name ChemicalH2N N N StructureN Standard RT (min) Sample RT (min) 1 Biopterin OH OH 3.65 3.68 N OH H2N N ON N OH N HO O 12 SepiapterinBiopterin O N O 3.654.16 3.684.17 OOHH H2N N N N N 11 Biopterin H2N NHON N O 3.653.65 3.683.68 2 2 SepiapterinSepiapterin N H H OH 4.164.16 4.174.17 2 Sepiapterin H2N N N OH 4.16 4.17 H NN OHN N O OH H2N2 N N 2 Sepiapterin H2N N H N H 4.16 4.17 H HN OH H2N N ON N O 2 Sepiapterin HO H OH 4.16 4.17 N OH H2N N ON N O 2 Sepiapterin N H O H OH 4.16 4.17 23 7,8-dihydrobiopterinSepiapterin O N OH 4.164.74 4.174.73 O OHOH H2N N N N N 2 Sepiapterin H2N NHON NH OH 4.16 4.17 3 7,8-dihydrobiopterin N H H OH 4.74 4.73 3 7,8-dihydrobiopterin H N N N OH 4.74 4.73 2H NN N N OH H2N2 HNO NH OH 3 7,8-dihydrobiopterin H2N NHH NHH 4.74 4.73 H HN OH H2N N N N 33 7,87,8-dihydrobiopterin-dihydrobiopterin OH H OH 4.744.74 4.734.73 H OH H N N N 2 N O OH 3 7,8-dihydrobiopterin HOO H OHOH 4.74 4.73 4 5,6-dihydrobiopterin O NHH OHOH 5.20 5.19 H N OH H2N N N N 3 7,8-dihydrobiopterin 2 NNHO HN OH 4.74 4.73 4 5,6-dihydrobiopterin N H H OH 5.20 5.19 4 5,6-dihydrobiopterin H N N N OH 5.20 5.19 2H NN N N OH 4 5,6-dihydrobiopterin H2N2 NOH NH OH 5.20 5.19 4 5,6-dihydrobiopterin H2N NHH HN 5.20 5.19 H ON OH H2N N ON N OH 4 5,6-dihydrobiopterin H H N 5.20 5.19 N NO OH 5 Pterin H2N N ON ON OH 6.21 6.20 H H N 4 5,6-dihydrobiopterin N N 5.20 5.19 H2N NNON N OH H2N N NN HN 455 5 5,6-dihydrobiopterinPterinPterin H 5.206.216.21 5.196.206.20 5 PterinPterin N OH 6.216.21 6.206.20 H NHHN2NNN ONNN NN 2 H2NO N H NOH 5 Pterin 2 H HH 6.21 6.20 HN N H2N N ON N 5 Pterin HN O H OH 6.21 6.20 6 6R-tetrahydrobiolumazine O H OH 6.63 6.66 H N OH O H2NN N ONNN N HNHNO HN OH 5 6 6R-tetrahydrobiolumazinePterin HN H H N 6.216.63 6.206.66 66 66RR--tetrahydrobiolumazinetetrahydrobiolumazine N OH 6.636.63 6.666.66 OH2N NN N N N OH 5 Pterin OHN NO HN OH 6.21 6.20 6 6R-tetrahydrobiolumazine O NH H HN H 6.63 6.66 H NH NH N OH OHN2 N N HN O O H OHOH 67 6RS-tetrahydrobiolumazine OH H H OH 6.637.77 6.667.82 O NH OHOH O N NH N OHNHNON N OH 6 7 6R6S-tetrahydrobiolumazine-tetrahydrobiolumazine HN H H 6.637.77 6.667.82 77 66SS--tetrahydrobiolumazinetetrahydrobiolumazine N OHOH 7.777.77 7.827.82 OHON N N N N OH OHN HNO HN OH 67 6RS-tetrahydrobiolumazine O ONH H HN H OH 6.637.77 6.667.82 H HN OH O N N OHN OHN HN OH 7 6S-tetrahydrobiolumazine N HO H OH 7.77 7.82 O H OHOH O N HN OH 8 6S-tetrahydrobiopterin H NHN NO NN OH 13.22 13.24 7 6S-tetrahydrobiolumazine 2 NNOH NH OH 7.77 7.82 8 6S-tetrahydrobiopterin N H HN OH 13.22 13.24 OHN N N OH 8 8 6S6S-tetrahydrobiopterin-tetrahydrobiopterin H NN N N OH 13.2213.22 13.2413.24 87 6S6-Stetrahydrobiolumazine-tetrahydrobiopterin H2N2 ONH NH OH 13.227.77 13.247.82 H2N N H NH H OH O HN HNN OH 8 6S-tetrahydrobiopterin H N N N 13.22 13.24 2 N OH OH OH H H H 8 6S-tetrahydrobiopterin N N OH 13.22 13.24 H2N N ON N N OH H HO O 99 5,6,7,85,6,7,8-tetrahydropterin-tetrahydropterin O HH 14.9914.99 15.01 15.01 8 6S-tetrahydrobiopterin N H NOH 13.22 13.24 H2N N N N N H2HN NNHON N 9 5,6,7,8-tetrahydropterin N H H OH 14.99 15.01 98 5,6,7,86S-tetrahydrobiopterin-tetrahydropterin H N N N N 14.9913.22 15.0113.24 2 H NN ON N H2HN2 HN N 9 5,6,7,8-tetrahydropterin H2N N H NH H 14.99 15.01 H HN H2N N ON N 9 5,6,7,8-tetrahydropterin H H 14.99 15.01 3.2. Mass Spectrometry Analysis N H2N N ON N H H 9 5,6,7,8-tetrahydropterin N 14.99 15.01 H2N N N N Although9 a few5,6,7,8 liquid-tetrahydropterin chromatography-tandem H massH spectrometry14.99 methods (LC-MS15.01 /MS) are H2N N N available to determine tetrahydrobiopterin [11–13], thereH H is shortage of spectrometric data acquired on high resolution MS systems. Therefore, LC-HRMSn (n = 1,2) spectra of each available impurity (1–3 and 5–9) were acquired and analyzed in order to investigate the fragmentation pattern. Mobile phase selection was made taking into account the pH used for the above described LC-UV detection (pH = 3.5). Considering that the use of a phosphate buffer is not compatible with ESI-MS ionization and that the analyzed compounds are small-size highly polar molecules, hydrophilic interaction liquid chromatography (HILIC) was selected to chromatographically resolve these compounds. Each standard was separately injected onto the column at a concentration level of 10 µg/mL and the full HRMS spectra obtained, were in all cases dominated by the [M+H]+ ion with the presence of a minor sodium adduct (Table S1, Supporting Information). Each standard was analyzed within 90 min from sample preparation in order to minimize degradation phenomenon that would result in the formation of oxidation products and a consequent dramatic signal decrease. Figure2 reports Extracted Ion Chromatograms (XICs) of the standards, obtained by selecting the relevant exact masses of each compound. Collision induced dissociation (CID) HR-MS2 spectra associated with each peak were also acquired (Figure S1, Supporting Information), and the elemental compositions for all the obtained fragment ions are reported in Table S2 (Supporting Information). Kuvan® and Diterin® tablets were then dissolved using the procedure reported in the Materials and Methods Section 2.2.2 and analyzed versus the standards under the same experimental conditions. Pharmaceutics 2020, 12, 323 6 of 10 Pharmaceutics 2020, 12, x 6 of 10

Figure 2. Extracted ion chromatogram (XIC) of compounds obtained by selecting exact masses of Figure 2. Extracted ion chromatogram (XIC) of compounds obtained by selecting exact masses of [M+H]+ and [M+Na]+ ions of each compound (see Table S1). [M+H]+ and [M+Na]+ ions of each compound (see Table S1). 3.3. Comparative Analysis of Kuvan® and Diterin® Tablets 3.3. Comparative Analysis of Kuvan® and Diterin® Tablets Having identified the nature of nine impurities present in sapropterin-containing tablets, we next employedHaving the above identified developed the nature HPLC of method nine impurities to the comparative present in analysissapropterin of Kuvan-containing® and Diterintablets,® we tablets.next employed Each product the was above analyzed developed at two HPLC different method time pointsto the ( T comparative= 0 and 6 months) analysis to quantify of Kuvan each® and singleDiterin impurity,® tablets. evaluate Each product the total was impurity analyzed amount at two and different its trend overtime thepoint timeframe.s (T = 0 and Representative 6 months) to examplesquantify of each the chromatogramssingle impurity,obtained evaluate atthe di totalfferent impurity times for amount different and products its trend are over reported the time inframe the . SupplementaryRepresentative Materials examples (Figures of the chromatograms S2 and S3). obtained at different times for different products are reportedTable2 reportsin the Supplement the percentageary concentrationMaterials (Figures of the S2 impurities and S3). for each sample, calculated as ( Ap/At) 100, whereTable 2A reportsp = area the of percentage the impurity concentration peak and A oft = thetotal impurities area of thefor impuritieseach sample, and calculated sapropterin. as (Ap × Data/At are) × 100, presented where asAp the = area mean of theSD impurity of at least peak three and independent At = total area measurements of the impurities carried and out sapropterin. for each ± sample.Data are Peak presented areas have as the been mean normalized ± SD of toat sapropterinleast three independent absorption on measurements the basis of the carried UV response out for each at 265sample. nm for Peak each compoundareas have (thebeen following normalizedε at to 265 sapropterin nm in 103 absorptionM 1 cm 1 onhave the been basis estimated of the UV 1 response= 14.8; × − · − 2 =at12.3; 265 3nm= 10.5;for each 4 = 10.2;compound 5 = 13.0; (the 6 and following 7 = 10.4;  at 8 and265 sapropterinnm in 103 × =M11.0;−1·cm 9−1= have10.8). been estimated 1 = 14.8; 2 = 12.3; 3 = 10.5; 4 = 10.2; 5 = 13.0; 6 and 7 = 10.4; 8 and sapropterin = 11.0; 9 = 10.8).

Pharmaceutics 2020, 12, 323 7 of 10

Table 2. Percentage concentration of the impurities for Kuvan® and Diterin® tablets at different times. Peak areas of ascorbic acid and sapropterin have been omitted. Data are presented as the mean SD of ± at least three independent measurements carried out for each sample.

Peak Kuvan® Diterin® Kuvan® Diterin® Number T = 0 T = 0 T = 6 T = 6 1 0.058 0.008 0.031 0.002 0.027 0.002 0.030 0.003 ± ± ± ± 2 0.035 0.006 0.037 0.003 0.221 0.012 0.169 0.007 ± ± ± ± 3 0.091 0.004 0.101 0.008 0.037 0.004 0.031 0.004 ± ± ± ± 4 0.051 0.002 0.032 0.003 0.176 0.009 0.050 0.005 ± ± ± ± 5 0.004 0.001 0.003 0.000 0.019 0.002 0.004 0.004 ± ± ± ± 6 0.090 0.007 0.011 0.001 0.135 0.009 0.010 0.001 ± ± ± ± 7 0.042 0.001 0.009 0.001 0.045 0.003 0.006 0.001 ± ± ± ± 8 0.018 0.001 Not detectable 0.026 0.003 0.015 0.003 ± ± ± 9 Not detectable 0.061 0.004 0.008 0.001 0.080 0.006 ± ± ± Total 0.389 0.0046 0.285 0.0039 0.694 0.0066 0.395 0.011 ± ± ± ±

4. Discussion Sapropterin is an effective agent in lowering blood hyperphenylalaninaemia (HPA) in patients with phenylketonuria (PKU) or BH4 deficiency, and, to date, no other natural or synthetic agent has demonstrated the same efficacy. Sapropterin is formulated for oral administration in tablets (or powder in sachets) that are dissolved in water prior to administration. The active pharmaceutical ingredient (API) is obtained synthetically through a somewhat complex route, with the main difficulty residing in controlling the configuration of three asymmetric centers. The processes giving rise to the API present in Kuvan® and Diterin®, enable the obtention of the 6R form with high purity, although very small amounts of the undesired 6S stereoisomer may be present. It is to be noted that the 6S form is not only inactive, but it may cause inactivation of phenylalanine hydroxylase. Sapropterin is a relatively unstable compound and its degradation chemistry is very complex, mostly ascribable to the sensitivity of side chain and ring moiety to oxidation. Tautomeric equilibria on the oxidized products render even more complex a comprehensive understanding of the exact formation and quantification of all the impurities. It has been estimated that in a sample of sapropterin left in the open, traces of biopterins 3 and 4 will start to appear after only 15 min incubation, and after 30 min, the amount of sapropterin is halved [11]. To minimize the impact of these reactions, sapropterin-based tablets contain the antioxidant ascorbic acid. As with any other medicine, sapropterin tablets can contain minor impurities deriving from their synthetic procedure as well as products formed upon degradation of the API itself. The aims of this work were: (a) to identify a separation method able to efficiently separate and quantify as many impurities as possible; (b) to apply this method to evaluate the impurities of commercially available sapropterin-containing tablets; and (c) to compare the impurity profile of generic and branded sapropterin-containing tablets, and finally evaluate them over time. To our knowledge, the method reported above, based on the use of an ion-exchange HPLC column, is one of the most efficient reported to date, as it enables the efficient separation and detection of nine impurities/degradation products from sapropterin-containing tablets. Data reported in Table2, evidence that the tablets contain an impurity percentage concentration ranging from 0.3% to 0.7%. If we had also taken into account the stabilizer ascorbic acid and the other excipients (mainly mannitol), the impurity percentage concentration would have been obviously further lowered. Figure3 schematically depicts the trend of impurity concentration for the examined products, when stored at 40 ◦C and 75% RH. As expected, an increase in the impurity level is observed over time for both tested commercial products, which can be clearly ascribed to an increase in the concentration of degradation products. The detailed trend of impurities is complex, and its full rationalization is extremely difficult; however, a more detailed analysis of the behavior of some selected impurities can better clarify this point. Pharmaceutics 2020, 12, x 8 of 10

Pharmaceutics 2020, 12, 323 8 of 10

0.8

0.7 Diterin® Kuvan® 0.6

0.5

0.4

0.3

0.2

0.1

Total impurities Percentage Total impurities 0 T = 0 T = 6 Months

Figure 3. Trend of total impurities concentration (Table2) for Kuvan ® and Diterin® at T = 0 and after

6 months at 40 ◦C and 75% RH (accelerated stability study). Figure 3. Trend of total impurities concentration (Table 2) for Kuvan® and Diterin® at T = 0 and after The6 concentrationmonths at 40 °C and of compound 75% RH (accelerated1 is practically stability study) constant. over the time and this could be explained by its origin as a synthetic impurity of the active principle sapropterin. The assignment of this peak fully The concentration of compound 1 is practically constant over the time and this could be supportsexplained this by hypothesis its origin as since a synthetic it has been impurit assignedy of the as active biopterin, principle a key sapropterin. intermediate The assignment in the syntheses of of ® ® sapropterin.this peak fully The supports average this level hypothesis of this impurity since it ha iss inbeen the assigned same range as biopterin for both, a Kuvankey intermediateand Diterin in . theFigure synthes4 reportses of sapropterin a likely,. The although average necessarily level of this simplified,impurity is in conversion the same range scheme for both that Kuvan could® help to rationalizeand Diterin® the. formation of the other compounds. Biolumazines (6 and 7) are the products of hydrolysisFigure of sapropterin4 reports a likely, (or ofalthough its epimer), necessarily resulting simplified, in the conversion substitution scheme of the that amino could grouphelp to on the pyrimidinerationalize ring the with formation a hydroxy of the group other andcompounds. subsequent Biolumazines tautomerism. (6 and Dihydrobiopterins 7) are the products3 andof 4 are regioisomerichydrolysis of oxidation sapropterin products (or of ofits sapropterinepimer), resulting [14], andin the the sub trendstitution of their of the concentrations amino group ison interesting. the pyrimidine ring with a hydroxy group and subsequent tautomerism. Dihydrobiopterins 3 and 4 are The concentration of 3 decreased over the time, probably because this compound was further oxidized regioisomeric oxidation products of sapropterin [14], and the trend of their concentrations is to sepiapterin (2), thus concurring to the marked increase at T = 6 of 2. In our experiments, at T = 6, interesting. The concentration of 3 decreased over the time, probably because this compound was ® sepiapterinfurther oxidized2 was the to majorsepiapterin impurity (2), thus in both concurring products, to the accounting marked increase for 0.169% at T in = Diterin 6 of 2. Inand our 0.221% ® ® in Kuvanexperiments,. In Diterinat T = 6, sepiapterinsepiapterin 2 was concentration the major impurity actually in both increased products, more accounting than 4-fold, for 0.169% meanwhile 7,8-dihydrobiopterinin Diterin® and 0.221% (3) in decreased Kuvan®. In 3-fold. Diterin It® sepiapterin was interesting concentration to note a thatctually the increased same trend, more even than if little bit more4-fold, accentuated, meanwhile 7,8 was-dihydrobiopterin found in Kuvan (3) ®decreased. On the 3 other-fold. It hand, was interesting direct oxidation to note tha of sapropterint the same to 4 causedtrend, an even increase if little of bit this more degradation accentuated, product was found at T in= 6Kuvan (2-fold®. On more the intenseother hand, as in direct Kuvan oxidation®), although, in turn,of sapropterin the oxidation to 4 cause of compoundd an increase4 ofcould this degradation provide a furtherproduct sourceat T = 6 of(2- pterinfold more (5) intense and biopterin as in (1). ® ImpurityKuvan5),showed although, a markedin turn, the increase oxidation at Tof= compound6 in Kuvan 4 could®, while provide only a afurther small source increase of pterin was noticed (5) in and biopterin (1). Impurity 5 showed a marked increase at T = 6 in Kuvan®, while only a small increase Diterin®. In Kuvan®, a parallel decrease of biopterin (1) at T = 6 was observed, therefore a conversion was noticed in Diterin®. In Kuvan®, a parallel decrease of biopterin (1) at T = 6 was observed, therefore of 1 into 5 may be hypothesized in this sample. a conversion of 1 into 5 may be hypothesized in this sample. A highly undesired impurity is (6S)-sapropterin epimer (8), which is capable of irreversibly inactivating the PAH enzyme [4]. Although both products contained a very little amount of this undesired side-product, after six months of storage in accelerated conditions, Diterin® demonstrated a roughly 50% inferior amount of 8 with respect to Kuvan®. Finally, 5,6,7,8-tetrahydropterin (9) could derive from a direct cleavage of the side chain of sapropterin and this impurity is present at T = 6 in higher concentrations in Diterin® than in Kuvan®. Grouping together all the impurities, we can observe that Kuvan® ranges from 0.389% (t = 0) to 0.694% (t = 6) (78% increase), while Diterin® ranges from 0.285% (t = 0) to 0.395% (t = 6) (39% increase).

Pharmaceutics 2020, 12, 323 9 of 10 Pharmaceutics 2020, 12, x 9 of 10

O OH H O OH N H HN hydrolysis N N OH O N N OH H H H2N N N side chain H H 6 cleavage Sapropterin O oxidation oxidation H N N O OH O OH H H2N N N N N H H N N 9 OH OH H2N N N H2N N N H H H 4 3 oxidative oxidation cleavage

oxidation oxidation

O O O N N N O OH N OH N N H2N N N H2N N N H H H OH 5 H2N N N 2 H side chain cleavage 1 Figure 4.4. ProposedProposed c conversiononversion scheme scheme for for the sapropterin degradation products detected in the HPLCHPLC-UV-UV analysis.analysis. 5. Conclusions A highly undesired impurity is (6S)-sapropterin epimer (8), which is capable of irreversibly inactivatingIn conclusion, the PAH data enzyme from an[4]. e Althoughfficient HPLC-based both products analysis, contained able toa very separate little andamount detect of ninethis undesiredimpurities /sidedegradation-product, products,after six months showed of an storage overall in lower accelerated level of co impuritiesnditions, Diterin/degradation® demonstrated products aand roughly higher 50% stability inferior of theamount sapropterin-containing of 8 with respect to generic Kuvan® tablets. Finally, Diterin 5,6,7,8® -formulation,tetrahydropterin which (9) mostly could deriverelate tofrom a lower a direct increase cleavage of impurities of the side2 chain, 4 and of6 sapropterin, compared and to Kuvan this impurity®. The lower is present increase at T of= 6 the in higherefficacy-reducing concentrations impurity in Diterin8, is also® than worthy in Kuvan of being®. G mentioned,rouping together because all although the impurities, it is numerically we can observeless significant, that Kuvan it is® potentially ranges from clinically 0.389% significant.(t = 0) to 0.694% (t = 6) (78% increase), while Diterin® ranges fromWe 0.285% are well (t = aware0) to 0.395% that a detailed(t = 6) (39% comparison increase) between. the two commercial products would require further investigation; it is, nevertheless, undisputable that both Kuvan® and Diterin® completely 5.conform Conclusion to the standard quality of the major regulatory authorities, even after six months storage in accelerated stability conditions. In conclusion, data from an efficient HPLC-based analysis, able to separate and detect nine impurities/degradationSupplementary Materials: products,The following show areed availablean overall online lower at http:level// ofwww.mdpi.com impurities/degradation/1999-4923/12 products/4/323/s1, andFigure higher S1: LC-HRMS stability/MS of the spectra sapropterin (CID mode)-containing obtained by gener selectingic tablets the [M Diterin+H]+ ion® formulation of each impurity, which/degradation mostly ® relatcompounde to a aslower precursor, increase Figure of S2:impurities HPLC profile 2, 4 and of Kuvan 6, comparedtablet at to T =Kuvan0 (top)®. and The T lower= 6 (bottom), increase Figure of the S3: HPLC profile of Diterin tablet at T = 0 (top) and T = 6 (bottom), Table S1: Exact masses, molecular formula (MF), efficacyRing Double-reducing Bond Equivalentsimpurity 8 (RDB), is also and worthy errors (of∆ppm), being measured mentioned for, [Mbecause+H]+ and although relevant it [M is +numericallyNa]+ ions of lesseach significant compound, contained it is potentially in Full scan clinically HRMS significant spectra, Table. S2: Exact assignment of the fragment ions contained in theWe LC-HRMS are well/MS aware spectra that of compounds a detailed 1–3 comparison and 5–9. Elemental between formulae the two of thecommercial mono-isotopic products ion peaks would (m/z) are reported with Ring Double Bond Equivalents (RDB) and errors (∆ppm). The most intense® fragment ion for® requireeach compound further is ininvestigation bold. ; it is, nevertheless, undisputable that both Kuvan and Diterin completely conform to the standard quality of the major regulatory authorities, even after six months Author Contributions: Investigation, E.S., F.V.; data curation, L.T., C.D.; writing—original draft preparation, storageO.T.-S., C.D., in accelerated L.T.; writing—review stability conditions. and editing, O.T.-S. All authors have read and agreed to the published version of the manuscript. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: LC- Funding: This research was funded by Dipharma SA, Chiasso, Switzerland by means of a Grant to O.T.-S. HRMS/MS spectra (CID mode) obtained by selecting the [M+H]+ ion of each impurity/degradation compound asConflicts precursor of,Interest: Figure S2Dipharma: HPLC profile S.A., of sponsor Kuvan® of tablet the study at T = and0 (top) producer and T = of 6 (bottom) Diterin®,, Figure provided S3: HPLC the products. profile Theof Diterin sponsor tablet had at no T role= 0 (top) in the and design T = 6 of (bottom the study,), Table nor inS1: the Exact collection masses, and molecular/or interpretation formula (MF), of the Ring data. Double Bond Equivalents (RDB) and errors (Δppm), measured for [M+H]+ and relevant [M+Na]+ ions of each compound contained in Full scan HRMS spectra, Table S2: Exact assignment of the fragment ions contained in the LC- HRMS/MS spectra of compounds 1–3 and 5–9. Elemental formulae of the mono-isotopic ion peaks (m/z) are

Pharmaceutics 2020, 12, 323 10 of 10

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