HPLC Determination of Purine Bases Possessing ... - Springer Link

Pharmaceutical Chemistry Journal

Vol. 38, No. 7, 2004

STRUCTURE OF CHEMICAL COMPOUNDS, METHODS OF ANALYSIS AND PROCESS CONTROL HPLC DETERMINATION OF PURINE BASES POSSESSING ANTIHERPETIC ACTIVITY (A REVIEW) P. T. Petrov,1 T. V. Trukhacheva,1 D. V. Moiseev,2 and A. I. Zhebentyaev2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 38, No. 7, pp. 44 – 53, July, 2004. Original article submitted December 9, 2003.

Aciclovir, or 9-(2-hydroxyethoxymethyl)-9H-guanine, is an acyclic analog of the natural nucleoside 2¢-deoxyguanosine. For many years, aciclovir occupies the leading position in the group of drugs used for the treatment of infectious diseases caused by herpes simplex virus (HSV) of types 1 and 2. The mechanism of the antiherpetic action of aciclovir is based on the inhibition of viral DNA synthesis. Penetrating into an HSV-infected cell, aciclovir is converted by viral thymidine kinase into aciclovir monophosphate. Under the 1 2

action of cell enzymes, this product is sequentially converted into acilovir diphosphate and triphosphate. Aciclovir triphosphate is capable of selectively blocking the assembly of viral DNA while practically not influencing the replication of human cell DNA. A disadvantage of aciclovir is the low bioaccessibility of the parent compound with respect to peroral administration. Various methods are used for increasing the bioaccessibility of this drug in various medicinal forms: (i) The formation of aciclovir esters [1 – 5]. Valaciclovir, representing aciclovir valine ester, is well absorbed in the organism upon peroral administration and metabolized with

“Belmedpreparaty” Joint-Stock Company, Minsk, Belarus. Vitebsk State Medical Institute, Vitebsk, Belarus

Valaciclovir

O N

HN

CH

C

N

N

O H2N

H2N

O

O

N

HO

N

HN

N

N

H2N

O

N

N

O H2N H3C

O

CH H3C

Famciclovir

OH

C

N

O O

CH3

OH

H3C

C

O

2'-Deoxyguanosine O

O N

HN H2N HO

N

N O

Aciclovir

O N

HN

N

N

H2N HO

H2N

N

N

HO

O

HO

N

HN

Ganciclovir

HO

Penciclovir

Structural formulas of aciclovir, 2¢-deoxyguanosine (a purine nucleotide involved in DNA synthesis), and their structural analogs.

391 0091-150X/04/3807-0391 © 2004 Springer Science+Business Media, Inc.

392

the formation of valine and aciclovir. Valaciclovir is available in the form of tablets and is used, as well as aciclovir, for the treatment of HSV-1, HSV-2, and varicella-zoster virus (VZV). (ii) The formation of stable water-soluble complexes. In particular, the solubility and, hence, bioaccessibility of aciclovir increases in a complex with b-cyclodextrin [6, 7]. (iii) The creation of aciclovir-containing liposomal compositions. Such preparations provide for an increase in the drug absorption through the skin [8 – 11]. (iv) The use of substances facilitating the penetration of drugs through mucous membranes. By forming complexes with acrylic acid and poly(ethylene glycol), it is possible to increase the penetration of aciclovir through biological membranes [12, 13]. (v) The controlled release of aciclovir from magnetically guided depot tablets. Using such tablets, it is possible to prolong the time of drug residence in the gastrointenstinal tract [14]. (vi) The use of electrophoresis. This provides a means of increasing the content of aciclovir in the focus of disorder [15]. Ganciclovir, or 9-(1,3-dihydroxypropoxymethyl)-guanine, is synthesized in the form of the sodium salt. This drug is more effective than aciclovir, but it is more toxic as well. In the organism, ganciclovir is also metabolized to triphosphate. Ganciclovir is used for the treatment of HSV-1, HSV-2, and cytomegalovirus (CMV) [16, 17] and is available (under the trade name cymevene) in the form of capsules and vials with lyophilized powder for injections. Famciclovir, or 6-deoxypenciclovir diacetyl ester, is metabolized in the organism with the formation of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)-guanine (penciclovir) and its triphosphate. The latter metabolite is capable of inhibiting the replication of viral DNA. Famciclovir and penciclovir inhibit the growth of HSV and VZV and are administered for the treatment of shingles and postherpetic neuralgia [16, 17]. Famciclovir is available in the form of tablets (famvir) and penciclovir, in the form of an ointment (vectavir). 1. PHARMACOKINETICS In the group of drugs under consideration, the pharmacokinetics was most thoroughly studied for aciclovir. The maximum concentration of aciclovir in the blood (6.7 – 20.6 mg/liter) was observed after intravenous injections in the course of treatment of immunodeficient states (2.5 – 15 mg/kg with 8-h interval). In the case of administration per os, the bioaccessibility of aciclovir amounts to 15 – 30% and the maximum drug concentration in the blood is observed 1.5 – 2.5 h after intake. For the ointment forms of aciclovir, the bioaccessibility is within 30 – 50%. The time of half-elimination (t1/2) after injections is 2 – 3 h. In the organism, aciclovir is distributed between many tissues

P. T. Petrov et al.

and fluids, including kidney, lungs, nervous tissues, liver, heart, cerebrospinal fluid, saliva, and tears [18]. Valaciclovir is well absorbed upon peroral administration and its bioaccessibility reaches about 54%, which is 2.5 – 3 times that of aciclovir. The half-elimination time determined in a group of volunteers upon a single administration of 100 – 1000 mg was t1/2 = 2.62 – 3.12 h [19]. The maximum concentration of valaciclovir in the blood serum upon administration in a single dose of 1000 mg was observed 1 – 3 h after intake and reached 27.1 ± 5.6 mg/liter [20]. In patients with liver disorders, the drug half-elimination time may increase up to 14 h. The bioaccessibility of ganciclovir after administration per os amounts to approximately 6% and the maximum drug concentration in the blood plasma (0.23 – 0.35 mg/liter) is observed 1.5 h after intake in a dose of 10 mg/kg [21, 22]. The half-elimination time was 4.5 h upon peroral administration (3000 mg/day) and 2 – 4 h upon intravenous injection (5 mg/kg) [23]. It was reported that, in patients with kidney disorders, the half-elimination time may increase up to 9 – 30 h [24, 25]. Famciclovir exhibits deacetylation and oxidation in the walls of intestine and in the liver with conversion into penciclovir [26]. The latter metabolite is characterized by high bioaccessibilioty (77%) and maximum concentration in the blood varying within 2.73 – 3.97 mg/liter 1 h after a single administration of 500 mg of famciclovir [27]. For a single administration of famciclovir in a dose of 125, 500, and 750 mg, the half-elimination time was 2.06 – 2.66 h [28]. 2. METHODS OF ANALYSIS The content of aciclovir and related drugs in ready-to-use preparations and their concentrations in the investigations of pharmacokinetics and optimum dosing were determined by various analytical techniques, including immunological methods and reverse-phase HPLC. Radioimmunological [29 – 33] and enzyme-linked immunoassay techniques [34, 35] are very sensitive but expensive methods involving laborious experimental stages and requiring the use of immune sera or monoclonal antibodies. Chromatographic methods are free of these disadvantages and are capable of separating antiviral agents in the analyzed medium even in cases when these compounds are structurally much like some endogenous substances. Chromatographic techniques are widely used for the determination of aciclovir and related drugs in the investigations of pharmacokinetics and in the course of therapeutic monitoring of the drug content in biological samples. These methods are stipulated in the normative pharmaceutical documentation for the drugs. These antiviral drugs can be also detected and analyzed by the method of capillary electrophoresis. High-performance capillary electrophoresis procedures have been developed and reported for the analysis of aciclovir [36 – 40] and

HPLC Determination of Purine Bases Possessing Antiherpetic Activity

related drugs [41] in pharmaceutical preparations and in biological fluids (sera, urine). Such systems are most frequently equipped with UV detectors, but fluorimetric (FL) and electrochemical detection schemes can be used as well. 3. CHROMATOGRAPHIC METHODS The samples of plasma, sera, urine, and tissues were analyzed for the content of aciclovir and ganciclovor by reverse-phase HPLC upon special sample preparation procedures, but experiments with direct sample introduction into a chromatograph have been described as well [42]. The samples of plasma taken upon aciclovir administration can be prepared for HPLC analysis in different ways, including deproteination by perchloric acid [43 – 46], trifluoroacetic acid [47], trichloroacetic acid [48], Ba-Al reagent [49 – 52], acetonitrile [53, 54], solid-phase extraction [53, 55 – 58], and ultrafiltration [59]. The introduction of an acid supernatant obtained upon deproteination by perchloric acid significantly reduces the working life of HPLC columns. In any case (even for a small volume of analyzed samples) only about 600 samples can be studied before substantial deterioration of the column efficiency [18, 45, 53]. In addition, the injection of acid supernatant leads to the appearance of numerous “late” peaks. These peaks additionally complicate the analysis and necessitate passage from the isocratic to the gradient elution regime for the removal of compounds responsible for the “late” peaks [43]. The solid-phase extraction [53, 55 – 58, 60, 61] eliminates these problems. Swart et al. [55] used for this purpose a Waters Sep-Pak Vac C-18 cartridge washed with 5 mM solution of sodium octanesulfonate (pH 2.85). Then, a sample with aciclovir was applied and the drug was eluted from the cartridge with the same solution at pH 8.5 (adjusted with 4 M NaOH/methanol solution in 4 : 1 ratio). The eluate was injected into a chromatograph. Poirier et al. [53] described the extraction procedure using a Waters Oasis HLB cartridge with a reverse-phase polymeric sorbent with lipophilic and hydrophilic functional groups. In the procedures of aciclovir determination in tissues and tears Rusak et al. [49, 50] precipitated proteins by treatment (i) with 0.7 M perchloric acid, after which the pH was adjusted to a neutral level by adding saturated KOH, or (ii) with a Ba-Al reagent (a mixture of 5% aluminum sulfate solution and 0.3 M barium hydroxide solution) for 15 min. Then, the samples were centrifuged for 20 min at 5000 rpm and aciclovir was determined in the supernatant by spectrophotometry at 254 nm with reference to 0.001% aqueos aciclovir solution. The Ba-Al reagent precipitated proteins more completely than perchloric acid (87% against 51%, respectively). During the HPLC analysis of aciclovir in suspensions, the presence of sodium carboxymethylcellulose (Na-CMC, ensuring increased viscosity of suspensions) leads to block-

393

ing of the column and hinders filtration, which reduces the working life of the column and requires frequent replacement of expensive filters. Epshtein [62] suggested adding ZnSO4 × 7H2O to the sample (4 : 1, w/w), which leads to virtually complete precipitation of Na-CMC in the form of floccules. The samples of plasma containing ganciclovir can be prepared for HPLC analysis using a number of methods, including acid deproteination [63 – 67], acetonitrile deproteination with chloroform extraction [68, 69], and ultrafiltration [65, 70]. Perchloric acid (0.8 M) can be added to a sample of blood serum in a volume ratio of 1 : 2, after which the supernatant is neutralized with 0.2 M phosphate buffer (pH 8) [65]. Chloroform extraction after the stage of deproteination with 50% trichloroacetic acid eliminates endogenous impurities frequently present in plasma extracts obtained using trichloroacetic and perchloric acids [66]. Cociglo et al. [68] used acetonitrile deproteination with chloroform extraction of the supernatant. Chloroform is preferred because of lower polarity and higher density [69]. Page et al. [65] prepared the samples using ultrafiltration via Centrifree (30,000 M) filters. Aciclovir has proved to be stable in samples of plasma stored for 4 weeks at –20°C. Nevertheless, it is recommended to collect and centrifuge the blood samples immediately, without storage at a low temperature (4°C) [45, 46]. No changes in the concentration of ganciclovir in working reference solutions were observed after a 1-month storage at 4°C. In samples of biological fluids, ganciclovir remained stable during storage –20°C for at least 6 months. However, the supernatant (pH 6.5 – 6.8) obtained upon precipitation with perchloric acid can be stored prior to analysis for no longer than 48 h at 4°C [53, 65 – 67]. Both aciclovir and ganciclovir can be used for the therapy of AIDS patients. Investigations of the effect of thermal pretreatment (40 – 70 min at 56 – 70°C) on the activity of drugs with respect to HIV virus revealed no significant differences between the heated samples and those kept at room temperature [53, 65]. 3.1. HPLC Determination of Aciclovir The main technological aspects of the HPLC analysis of aciclovir, including the column types and characteristics, elution conditions, and detection parameters are summarized in Table 1. Aciclovir is most frequently analyzed in the reverse-phase HPLC mode, but normal-phase regimes were reported as well [107, 115]. The analyses are usually performed at room temperature; higher temperatures (up to 40°C) are used for the analysis of liposomal preparations. The mobile phase is usually characterized by a high content of buffer (pH 2.5 – 3.5) and is supplied at a rate of 1.0 – 1.5 ml/min. Spectrophotometric (UV) detectors are usually tuned to a wavelength of 250 – 254 nm, whereas fluorimetric (FL) detectors operate at an excitation

394

P. T. Petrov et al.

TABLE 1. Main Parameters of HPLC Determination of Aciclovir Object

Plasma Liposomes

Column: type (length ´ diameter, particle size), temperature (°C)

Silica gel C-18 (300 ´ 4.6 mm, 10 mm) Spherisorb S5-ODS (250 ´ 4.6 mm, 2 mm), 40°C

C-8 (150 ´ 4.6 mm, 5 mm), precolumn C-8 (1 cm, 5 mm) Drug suspension Supercosil LC-18 (75 mm, 3 mm) Plasma

Plasma Serum, urine

Skin Plasma

Serum Eye tissues and tears Eye tissues and tears

Hypersil ODS (150 ´ 4.6 mm, 3 mm) Ultrasphere ODS RP (75 ´ 4.6 mm, 3 mm)

C-18 (250 ´ 4.6 mm, 5 mm) LiChrosorb RP-8 (250 ´ 4 mm, 7 mm), precolumn Perisorb RP-18 (30 – 40 mm) Techsphere 5 C-8 (100 ´ 4 mm) Silasorb C-18 (250 ´ 4 mm, 10 mm), 40 °C Silasorb C-18 (250 ´ 4 mm, 10 mm), 40°

Serum, sheep C-8 (150 ´ 4.6 mm) blood, saliva, urine Drug suspension Symmetry C-18 (75 ´ 4.6 mm, 3.5 mm), precolumn Symmetry C-18 (20 ´ 3.9 mm, 5 mm), 30 °C Urine, serum LiChrospher C-18 (250 ´ 4.6 mm, 10 mm), 35 °C Plasma, amnion- HP Agilent Eclipse XDB ic fluid, pla- C-8 (150 ´ 2.1 mm) centa, tissues

Skin and penetrating mixtures

LiChrospher Select C-8 (250 ´ 4 mm)

Mobile phase

Detector DetermiSample Flow rate, waveDetection nation volume, limit, threshold ml/min length l, ml ng/ml nm

Ref.

Methanol – 50 mM octanesulfonic buffer (pH 2.5), 8 : 92 (v/v) Methanol – 5 mM potassium dihydrophosphate (pH 3), 5 : 95 (v/v) + 7 mM hexylamine

1.5

254

100

—

20

[46]

1.3

254

10

—

1000

[90]

0.1 M acetate/citrate buffer (pH 3.5) – 3.7 mM octanesulfonic acid, 8 : 92 (v/v) and methanol

1.0

250

100

10 ng/ml

62

[53]

Acetonitrile – 100 mM glycine buffer (pH 2.3), 3 : 97 (v/v)

1.0

20

—

25

[74]

20 mM potassium dihydrophosphate (pH 3.5)

1.5

lex = 260 lem = 375 254

20

—

100

[45]

30 mM phosphate buffer (pH 2.1) + 5 mM sodium dodecylsulfate + 18% acetonitrile

1.5

lex = 285 lem = 380

20

—

[58]

Distilled water

1.2

254

50

0.12 mM in sera, 0.60 mM in urine —

8 in skin

[108]

1% acetonitrile + 20 mM sodium monohydrophosphate (pH 2.5)

1.2

lex = 270 lem = 380

50

30 ng/ml

—

[44]

1% orthophosphoric acid + 10 g/liter octanesulfonic acid 5 mM sodium afetate + 5 mM sodium dodecylsuylfate (pH 6.85) and 2-propanol, gradient: 2 – 20%/20 min 5 mM sodium afetate + 5 mM sodium dodecylsuylfate (pH 6.85) (1) and 2-propanol (2); schedule: 1 – 8 min, 100% (1); 8 – 21 min, 85% (1); above 21 min, 80% (1) 0.02 M HClO4 (pH 2.0)

1.0

254

20

500 ng/ml

—

[101]

2.0

254

—

—

—

[49]

2.0

—

10

—

—

[50]

1.5

260 lem = 376

—

—

[75]

Acetonitrile – methanol – 10 mM potassium duhydrophosphate (pH 4.0), 25 : 25 : 50 (v/v)

1.0

256

20

10 – 20 ng/ ml (UV); 50 pg/ml (FL) —

—

[62]

5 mM sodium dodecylsulfate – 2-propanol – acetic acid (87.5 : 10 : 2.5, v/v)

1.5 (for urine) 2.0 (for serum) 0.2

253

—

—

—

[109]

254

—

—

250

[110]

252

—

6 ng/ml

50

[111]

Plasma (amnionic fluid), 10 mM acetate/citrate buffer – 3.7 mM octanesulfonic acid, 87.5 : 12.5 (v/v) tissues, 30 mM acetate/citrate buffer – 5 mM octanesulfonic acid (pH 3.08), 99 : 1 (v/v) Acetonitrile – 50 mM ammonium acetate (pH 6.5), 1 : 99 (v/v)

250

—

—


395

TABLE 1. (C o n t i n u e d ) Object


Plasma

LiChrospher 100 C-8

Plasma

YWG C-18 H37 (300 ´ 3.9 mm, 10 mm) Bondapak NH2 (30 ´ 4.6 mm) Nova Pak C-18 (150 ´ 3.9 mm, 4 mm), precolumn Perisorb RP-18 (30 mm, 40 mm)

Serum, urine Plasma

Serum

RP-8 (125 ´ 4.5 mm)

Serum, plasma

Separon SGX (150 ´ 3.2 mm, 5 mm)

Plasma

Nucleosil 120 3C-18 (80 ´ 4 mm, 4 mm), 30 °C

Plasma

Bondapak C-18 (300 ´ 3.9 mm, 10 mm) Bondapak C-18 (300 ´ 3.9 mm)

Capsules, ointments, injection solutions Serum Nucleosil C-18 (200 ´ 4 mm, 5 mm) Plasma Spheron Micro 300 (150 ´ 3.2, 12.5 mm), precolumn Spheron Micro 300 (16 mm) Biological fluids Supercosil LC-18 (150 ´ 4.6, 5 mm), precolumn Pelliguard Plasma YWG C-18 H37 (200 ´ 5.0, 10 mm) Serum PRP-1 (150 ´ 4.2, 10 mm)

Plasma

Bondapak C-18 (300 ´ 3.9, 10 mm)

Ointments

Nucleosil C-18 (250 ´ 4 mm, 5 mm) Supelco LC-18 (250 ´ 4.6 mm, 5 mm)

Tablets, ointments

Parent C-18 (300 ´ 4.2 mm) 1 substance1, C-18 (250 ´ 4.6 mm) 2 – 6 tablets2, suspensions3, ointments4, injections5, capsules6

Mobile phase


Ref.

18% acetonitrile – 5 mM sodium dodecylsulfate, phosphate buffer (pH 2.6) methanol – water, 5 : 95 (v/v)

—

250 – 260

—

6 ng/ml

20

[112]

2.0

254

—

20 ng/ml

200

[56]

Acetonitrile – water, 80 : 20 (v/v)

—

—

—

—

—

[113]

Methanol – 10 mM sodium monohydrophosphate (with 10 mM sodium octanesulfate, pH 2.8), 7 : 93 (v/v)

1.0

250

130

10 ng/ml

5

[55]

Methanol – 50 mM octanesulfonic acid (in 0.1 M phosphate buffer, pH 3.0), 1):)19 (v/v) 50 mM phosphate buffer with 10 mM hexadecyltrimethylammonium bromide (pH 2.05) 20 mM perchloric acid

1.0

254

—

50

500

[59]

lex = 285 lem = 370

10

80 ng/ml

7% methanol with 5 mM sodium octanesulfonate

1.0

lex = 260 lem = 375 254

3% acetonitrile with 10 mM potassium dihydrophosphate

2.0

252

Methanol – water, 5 : 95 (v/v)

1.0

250

0.1 M orthophosphoric acid + 0.1 M sodium sulfate (pH 1.8)

1.0

lex = 285 lem = 370

5 mM sodium acetate + 2.5 mM sodium pentanesulfonate (pH 6.5 adjusted with NaOH)

1.2

254

7% methanol + 2.5 mM sodium heptanesulfonate + 5 mM sodium acetate

—

Methanol + 0.1 M HCl + 0.02 sodium heptanesulfonate + 0.25 M NaCl, 1 : 1 : 2 : 6 (v/v) Acetonitrile + 10 mM potassium dihydrophosphate, 1 : 999 (v/v) with 0.9 g/liter heptanesilfonic acid glacial acetic acid – water, 0.25 : 99.75 (v/v) 10 mM sodium dihydrophosphate (pH 3.5 adjusted with phosphoric acid) and acetonitrile; gradient: 5 – 25% actetonitrile/20 min 20 mM acetic acid

[114]

5.5 ng/ml

10.3

[43]

100 ng/ml

500

[48]

30

—

—

[116]

25

300

1250

[117]

1 ng/ml

1.0

[42]

20

—

—

[51]

254

5

5

750

[52]

1.5

254

—

12

120

[47]

—

254

—

—

250

[54]

1.5

254

20

—

—

[84]

1.0

254

20

—

—

[81, 83]

3.0 1, 3, 4 1.0 5, 1.5 2, 6

254

201–4, 6, 50 5

—

—

[85]

1.5

396

P. T. Petrov et al.

TABLE 1. (C o n t i n u e d ) Object


Parent substance C-18 Silica (100 ´ 4.6 mm, 3 mm)

Plasma, amnion- PH Agilent Eclipse XDB ic fluid, pla- C-8 (150 ´ 2.1 mm), centa Phenomenex C-18


Mobile phase

6.0 g sodium dihydrophosphate + 1.0 g sodium decanesulfonate in 900 ml water (pH 3.0 adjusted with phosphoric acid), 40 ml acetonitrile, water to 1000 ml 30 mM acetate/citrate buffer (pH 3.1) and methanol; gradient elution

wavelength of lex = 260 – 285 nm and an emission wavelength of lem = 375 – 380 nm. In recent years, there were cases of using mass-spectrometric [71] and pulsed ammetric [72] detectors. The accuracy and detection limits of HPLC analysis depend on the pH of the mobile phase. At pH 3.0, the retention time of aciclovir is 5.3 min; by adjusting the mobile phase acidity within 2.75 – 3.25, it is possible to increase the retention time by 10% and decrease it by 12% [53]. Poirer et al. [53] performed the analyses at pH 3.0 and reached a determination limit of 62.5 ng/ml at a detection threshold of 10 ng/ml. An increase in the sensitivity can be achieved by using a fluorimetric detector instead of a UV detector, but the fluorescent response of aciclovir is strongly pH-dependent (significantly increases at pH below 2) [73]. Therefore, only strongly acidic mobile phase may provide for an increase in the detection threshold, but very low pH (~1.5) may significantly deteriorate the resolution of the analytical column [43]. Using a mobile phase adjusted at pH 2.5 with 60 – 62% perchloric acid, it is possible to reach a detection threshold of 30 ng/ml with a fluorimetric detector, which is quite satisfactory for the investigations of pharmacokinetics. A system with fluorimetric detector and a mobile phase adjusted at pH 2.3 was reported to provide for a detection limit at 10 ng/ml [74]. The proposed procedure can be successfully used for studying the parmacokinetics of various medicinal forms of aciclovir. Most of the HPLC procedures developed for aciclovir determination did not stipulate the use of internal standards, which reduced the accuracy and reproducibility of analysis [45]. It was suggested [53] to use guanosine, a compound structurally close to aciclovir and having a retention time of 7.8 min (against 5.3 min for aciclovir), as the internal standard. Endogenous guanine is usually present below a detectable level (10 ng/ml) in both control solutions and samples of blood serum taken from patients. A very simple and time-consuming procedure for the determination of aciclovir was proposed by Bangaru et al. [46]. Using this method, aciclovir was reliably determined in the plasma of healthy volunteers upon a single peroral administration in a dose of 400 mg. A low determination limit

Ref.

2.0

254

20

—

—

[80]

0.15 – 0.25

254

—

—

100

[72]

(~20 ng/ml) allowed the pharmacokinetic parameters of absorption to be determined up to the last discretization point. The method is characterized by good reproducilibity; the calibration curves are linear in the range of aciclovir concentrations from 0.02 to 5 mg/ml. According to Testereci et al. [75], using 0.02 M perchloric acid solutiuon (pH 2.0) as the mobile phase allows the detection threshold for aciclovir to be reduced to 10 – 20 ng/ml with a UV detector and to 50 pg/ml with a fluorimetric detector. The proposed procedure was used for determining aciclovir in the blood, saliva, and urine of experimental animals. The standard procedure for determining aciclovir and guanine (the main technological impurity appearing in the course of synthesis and the product of decomposition of the parent compound) is described in the normative documentation for aciclovir preparations. According to this, the impurities are determined by TLC (76 – 80] or HPLC [80 – 89]. The high pK a of guanine complicates reverse-phase HPLC analysis in columns with silica gel based sorbents. There are four main approaches to solving this problem: (i) using ion-pair reagents (this is advantageous for technological quality control, but the analysis of unknown samples involves difficulties in establishing the amount of ion-pair reagent in the mobile phase); (ii) conducting the analysis at neutral pH in inactivated silica gel columns (this requires a large amount of the organic component in the mobile phase and increases the cost of the HPLC procedure); (iii) using polymer-based columns; (iv) using silanol-masking agents binding to the immobile phase of the matrix and ensuring better protection of silanol groups. Caamano et al. [90] showed that using hexylamine as the silanol-masking agent increases the hydrophobic properties of the immobile phase. Sodium deoxycholate (surfactant), which solubilizes the phospholipid bilayer and releases the drug from the liposomal matrix, is not eluted under the HPLC conditions employed and, hence, does not interfere with the analysis for aciclovir. 3.2. HPLC Determination of Valaciclovir HPLC procedures developed for determining aciclovir are usually not intended for detecting its precursor


valaciclovir. HPLC determination of valaciclovir was described in [91 – 93]. Weller et al. [92] determined valaciclovir in the presence of aciclovir by HPLC with elution in a special gradient regime. A simple, specific, and rapid HPLC procedure with isocratic elution for the simultaneous determination of valaciclovir and aciclovir in biological fluid was developed by Pham-Huy et al. [93]. Quantitative analyses were performed using 1-methylguanine as the internal standard. The samples of sera were deproteinated by perchloric acid. Urine samples were diluted with a mobile phase prepared by mixing acetonitrile with 0.025 M monoammonium phosphate buffer (2 : 98, v/v), with pH 4.0 adjusted with a 10% aqueous phosphoric acid solution. The analyses were performed on a Symmetry Shield RP-C8 (250 ´ 4.6 mm, 5 mm) column in the HPLC system with a protective (20 ´ 3.9 mm) column. The mobile phase flow rate was 1.0 ml/min; the sample and internal standard volumes were 50 ml; and the UV detector was tuned to 254 nm. The detection thresholds for aciclovir and valaciclovir were 50 and 75 ng/ml, respectively. The standard calibration plots for the samples of blood serum, urine, and dialyzed fluids were linear in the range of drug concentrations from 0.5 to 20.0 mg/ml. The reproducibility of measurements with the internal standard was 94 – 95%. Savaser et al. [94] also used HPLC with an internal standard and reached a determination threshold of 0.14 ng/ml for valaciclovir in the blood serum. The calibration plot was linear in the interval from 5 to 20.000 ng/ml. The analyses were performed with a Waters Spherisorb C-18 (250 ´ 4.6 mm, 5 mm) column eluted with a mobile phase representing a mixture of acetonitrile, methanol, and 0.067 M potassium dihydrophosphate (27 : 20 : 53) at pH 6.5 (adjusted with 3 M NaOH solution). The eluent flow rate was 0.75 ml/min; the UV detector was tuned to 244 nm. 3.3. HPLC Determination of Ganciclovir Ganciclovir is determined by reverse-phase HPLC at room temperature or 30 – 40°C using C-18 or C-8 columns with a system of precolumns. The mobile phase typically contains a high percentage of buffer at pH 2.1 – 6.6 and is supplied at a rate within 1.0 – 2.0 ml/min. The HPLC systems are equipped with spectrophotometric detectors tuned to a wavelength of 254 nm or with fluorimetric detectors operating at lex = 278 nm and lem = 380 nm. The HPLC determination of ganciclovir is complicated by the fact that the chemical structure of its molecule, which is a guanine analog, is very close to the structure of the endogenous compound. In addition, being highly polar, ganciclovir molecules are weakly retained on nonpolar immobile phases of C-18 and C-8 columns. The selectivity of analyis of biological samples can be increased by adding ion-pair reagents or organic modifiers (e.g., triethylamine) to the mobile phase. Such additives decrease the polarity of ganciclovir, but this is achieved at the expense of rapid deterioration of the column, the appearance of foreign peaks,

397

baseline shift, and the aggregation and deposition of polymers or macromolecules entering into the composition of microparticles in dosed medicinal forms. The potential interference of endogenous substances during the analysis of blood plasma can be eliminated in HPLC systems using electrospray ionization and mass-spectrometric detection [95]. The role of the internal standard in the quantitative analysis can be played by aciclovir [66, 68, 96] or 9-methylxanthine [64, 97]. Merodio et al. [96] suggested using ammonium acetate in the mobile phase without an ion agent or any other organic modifier. In addition, they used a column with LiChrospher C-8, which is a somewhat more polar immobile phase than C-18. Campanero et al. [66] introduced triethylamine into the mobile phase in order to improve the shape of the chromatographic peaks. Acting as an organic modifier, triethylamine interacts with free silanol groups in the immobile phase, thus inactivating these groups and eliminating peak “tails.” In the mobile phase with pH 6.6, ganciclovir occurs in a neutral form and can be analyzed by reverse-phase HPLC. Using buffered mobile phases with pH 2.4 [65] and 2.5 – 2.9 [98], it is possible to perform more than 3000 analyses without significant deterioration of the column. The retention time depends on the characteristics of the mobile phase and, in some systems, on the content of acetonitrile in the mobile phase. For example, in the aforementioned procedure [96], the retention time increased from 3.5 to 5.2 min when the acetonitrile content was reduced from 5 to 1%. In some other cases, the retention time of ganciclovir varied within the interval from 4 to 9 min. The time of analysis may also depend on the separation procedure and the flow rate. The detection threshold for ganciclovir varies from 3 to 30 ng/ml and the determination limit, from 10 to 50 ng/ml. For determining ganciclovir in blood plasma, the latter limit can be reduced by increasing the injected sample volume and/or the aliquot used at the stage of extraction and isolation. The reverse-phase HPLC procedure [66] can be used for determining ganciclovir in the blood plasma, therapeutic drug monitoring, and studying the pharmacokinetics of ganciclovir in healthy volunteers and in patients with renal dysfunction and upon kidney transplantation. The working range of concentrations is 1 – 200 ng/ml, the linearity is ensured in the interval from 0.05 to 10 mg/ml (UV detector), and the reproducuibility is 95 ± 3.26%. Koel and Nebinger [70] showed that a fluorimetric detector provided for a 5-fold decrease in the determination limit as compared to that achieved with the UV detector. The HPLC analyses were performed on a LiChrospher RP-8 (125 ´ 4.6 mm, 5 mm) column eluted with a mixture (1 : 19) of methanol and 0.05 mM octanesulfonic acid solution in 0.1 M phosphate buffer (pH 3). The mobile phase flow rate was 1 ml/min; the spectrophotometric detector was tuned to 254 nm, and the fluorimetric detector was operating at lex = 285 nm and lem = 380 nm. The linearity of detection was ensured in the intervals from 0.05 to 100 mg/liter (UV

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detector) and 0.01 to 50 mg/liter (fluorimetric detector) at a reproducibility of 98 – 100%. The HPLC procedures described by Boulieu et al. [64, 97] involve an internal standard (9-methylxanthine) and allow detecting 0.5 ng of ganciclovir using a Hypersil ODS (150 ´ 4.6 mm, 5 mm) column eluted with an 0.02 M potassium dihydrophosphate solution at pH 3.5 and 5.25 [97] and pH 5.25 [64]; the eluate flow rate was 1.5 ml/min and the UV detector was tuned to 254 nm. Chu et al. [98] decreased the detection threshold to 0.04 – 4.00 mg/ml by using the fluorimetric detector. The determination threshold was also sufficiently low (0.05 mg/ml) to provide for the possibility of pharmacokinetic investigations. A relatively small sample volume (25 ml) allowed the procedure to be used for studying the drug pharmacokinetics in children, with the reproducibility varying within 94.0 – 110%. In order to further reduce the detection threshold Tsuchie et al. [99] suggested converting ganciclovir into a fluorescent compound by reaction with phenylglyoxal [100]. The subsequent analyses were performed at 20 – 25°C on a Develosil (150 ´ 4.6 mm, 5 mm) column eluted with a mixture (18 : 82, v/v) of acetonitrile with a 1 mM phosphate buffer (pH 6.2) at a flow rate of 1.0 ml/min; the fluorimetric detector operated at lex = 365 nm and lem = 512 nm. Bleyzac and Boulieu [63] determined ganciclovir in myocardial tissue using a Hypersil ODS (150 ´ 4.6 mm, 5 mm) column eluted with an 0.02 M potassium dihydrophosphate solution (pH 3.5) at a flow rate of 1.5 ml/min, with a UV detector tuned to 254 nm. The determination and detection limits were 40 mM and 2 pM, respectively; the results were reproduced at a level of 101 ± 2%. VcMullin et al. [101] developed a method for the separation and simultaneous determination of aciclovir and ganciclovir. The analyses were performed on a Techsphere 5 C-8 (100 ´ 4 mm) column eluted with 1% orthophosphoric acid solution containing 10 g/liter of octanesulfonic acid. The mobile phase flow rate was 1 ml/min; the spectrophotometric detector was tuned to 254 nm. The samples were deproteinated with 7% perchloric acid. The injected volume of supernatant was 20 ml. The detection thresholds for aciclovir and ganciclovir were 500 and 300 ng/ml, respectively. The degree of drug extraction from blood serum for both antiviral drugs was about 100% in the range of concentrations from 0 to 100 mg/ml. The coefficient of correlation between the peak height and the drug concentration for aciclovir and ganciclovir in the blood serum was r = 0.994 and 0.999, respectively. The error of drug determination in the interval of concentrations from 1 to 9 mg/ml was 0 – 3.2% for aciclovir and 1.1 – 6.7% for ganciclovir. 3.4. HPLC Penciclovir

Determination

of

Famciclovir

and

Penciclovir is the major metabolite found in blood plasma and urine upon the administratuion of famciclovir;

the minor metabolite fractions include penciclovir monoacetylate and 6-deoxypenciclovir monoacetylate. The concentrations of penciclovir and 6-deoxypenciclovir in samples of plasma and urine were determined by reverse-phase HPLC [102 – 104]. The proposed methods were developed for the investigation of famciclovir pharmacokinetics. The limits of quantitative determination of penciclovir and 6-deoxypenciclovir in samples of blood plasma were 0.2 and 0.4 mg/ml and in urine, 10 and 20 mg/ml, respectively. All procedures were linear in the interval of concentrations from 1 to 80 mg/ml. The analyses are complicated by the fact that some metabolites are eluted together with the penciclovir fraction. In order to solve this problem, Hsu et al. [41] developed an HPLC procedure using reverse-phase YMC AQ C-18 (250 ´ 4.6 mm, 5 mm) column. The method has proved to be sufficiently accurate and well reproducible, the only disadvantage being that a low-ionic-strength solvent necessary for good resolution increased the peak width and prolonged the separation time to 22 min. The samples were eluted in the isocratic regime with a methanol – water (5 : 95, v/v) mixture containing 23 mM potassium phosphate buffer (pH 7.0). The mobile phase flow rate was 1.0 ml/min, the column temperature was 30°C, and the detection threshold was 0.5 mg/ml at a sample volume of 20 ml. The time of analysis could be significantly reduced (to 7 min) using a capillary electrophoresis technique. For monitoring the purity of penciclovir in the course of production, Goodwin [105] suggested to perform a single-stage gradient elution instead of three stages of isocratic elution. The analyses were performed on a Spherisorb ODS (250 ´ 4.6 mm) column eluted at a mobile phase flow rate of 2 ml/min, with UV detection at 254 nm. The mobile phase was either a 0.1 M solution of sodium dihydrophosphate with pH 3.7 (A) or a mixture of solution A with acetonitrile, 79 : 21 (B). The gradient elution schedule was as follows: 0 – 5 min, 100% A; 5 – 35 min, 100 to 65% A; 35 – 50 min, 35 to 100% B; above 50 min, 100% B. Using this procedure, it is possible to separate up to 16 potential impurities in penciclovir. 4. CONCLUSIONS The appearance of aciclovir – the first specific antiviral drug – in 1977 marked a considerable progress in antiviral chemotherapy. More than two decades layer, the role of acviclovir as an effective antiherpetic agent is now clearly established. This drug occupies a leading position among preparations intended for the prophylaxis and treatment of HSV and zoster infections. Treatment with aciclovir and related drugs is especially important in the immunosuppressed population, in view of the rapid and global spread of AIDS and the increasing practice of transplanting bone marrow and other organs.


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